Compositions for Preventing Cardiac Arrhythmia

ABSTRACT

Disclosed herein are compositions and methods for treating or preventing a cardiac arrhythmia in a subject.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/480,140, filed on May 24, 2012, which is a Continuation-In-Part ofU.S. application Ser. No. 12/707,427, filed on Feb. 17, 2010, whichclaims the benefit of U.S. Provisional Application No. 61/153,402, filedon Feb. 18, 2009; U.S. Provisional Application No. 61/491, 723, filed onMay 31, 2011; and U.S. Provisional Application No. 61/650,911, filed onMay 23, 2012; which are hereby incorporated by reference in theirentireties.

BACKGROUND

Cardiac arrhythmias present a significant health problem. Cardiacarrhythmias include, but are not limited to, ventricular tachycardias,supraventricular tachycardias, and atrial fibrillation. Of these, atrialfibrillation is the most common cardiac arrhythmia. It has beenestimated that over one million people in the United States alone sufferfrom atrial fibrillation. The incidence of atrial fibrillation isexpected to increase over the next several decades as populations in theUnited States and Europe trend older because atrial fibrillation tendsto become more common with increasing age.

Arrhythmias after cardiac surgery are a major cause of morbidity andmortality. Tolerability of arrhythmia is less in the postoperativeperiod than for similar arrhythmias in the preoperative period.Hemodynamic instability is more likely due to the possibility ofmyocardial dysfunction. Cardiopulmonary bypass; injury to the conductionsystem during surgery; and metabolic and electrolyte abnormalities,especially hypokalemia and hypomagnesemia, contribute to the increasedincidence of postoperative arrhythmias. Stress of the surgery withenhanced sympathetic tone and use of inotropic support are addedfactors. Delayed arrhythmia can occur due to scar-related re-entry.

Atrial fibrillation can be treated with medication intended to maintainnormal sinus rhythm and/or decrease ventricular response rates.Specifically, many of the past attempts have been confined topharmacotherapy, radiofrequency ablation, use of implantable devices,and related approaches. While drug therapy remains a popular route forreducing some arrhythmic events, there has been recognition thatsystemic effects are often poorly tolerated. Moreover, there is beliefthat proarrhythmic tendencies exhibited by many drugs can increasemortality in many situations. It would be desirable to have moreeffective compositions and methods for treating or preventing cardiacarrhythmias.

The invention generally relates to sterilized, acellular extracellularmatrix compositions and methods of making such compositions for use intreating or preventing cardiac arrhythmias. More particularly, theinvention relates to methods of contemporaneously sterilizing anddecellularizing extracellular matrix compositions, as well as thesterilized, acellular compositions resulting from such methods for usein subjects who have undergone heart surgery or had a myocardialinfarction to treat or prevent cardiac arrhythmia.

Conventional techniques for sterilizing tissue compositions often alterthe properties of the tissue compositions and/or damage importantcomponents of the tissue compositions, such as growth factors.Consequently, these conventional sterilization techniques often rendertissue compositions unfit for their intended purposes. For example,ethylene oxide is a toxic, mutagenic, and carcinogenic substance thatcan weaken tissue compositions, reduce the growth factor content oftissue compositions, and denature proteins within tissue compositions.Similarly, conventional steam sterilization techniques are incompatiblewith the biopolymers of tissue compositions, and gamma radiation causessignificant decreases in the integrity of tissue compositions. Althoughthere are known techniques for sterilizing tissue compositions withoutaltering the properties of the tissue compositions, many of thesetechniques, such as anti-bacterial washes, often fail to completelysterilize the tissue compositions and/or leave residual toxiccontaminants in the tissue compositions.

Additionally, when tissue compositions are designed for implantationwithin the body of a subject, the tissue compositions must often beexposed to a separate, time-consuming decellularization process. Thisdecellularization process is intended to remove cells from the tissuecompositions, thereby decreasing the likelihood that the subject'simmune system will reject the implanted tissue compositions and/orgenerate a significant inflammatory response. However, conventionaldecellularization techniques merely decellularize portions of the tissuecompositions such that native cells remain in the tissue compositionsfollowing the decelluarization process.

U.S. Pat. No. 7,108,832 (the '832 patent), which is assigned toNovaSterilis, Inc., discloses a method that sterilizes various materialsthrough the use of supercritical carbon dioxide. However, as with otherknown sterilization methods, tissue compositions that are sterilizedusing the process disclosed in the '832 patent must be exposed to aseparate decellularization process, as described above.

Accordingly, there is a need in the art for a method of sterilizing anddecellularizing a tissue composition, such as an extracellular matrixcomposition. More particularly, there is a need in the art for a methodof (a) sterilizing a tissue composition while maintaining the nativeproperties of the tissue composition and (b) decellularizing the tissuecomposition such that the tissue composition is acellular. There isstill a further need for a method of enhancing the incorporation ofadditives into a tissue composition during sterilization and/ordecellularization of the tissue composition for purposes of treating orpreventing cardiac arrhythmia.

SUMMARY

In accordance with the purpose of this invention, as embodied andbroadly described herein, this invention relates to compositions andmethods for treating or preventing cardiac arrhythmia in a subject.

This invention also relates to methods of sterilizing anddecellularizing an extracellular matrix (ECM) material for use intreating or preventing cardiac arrhythmia in a subject who has undergoneheart surgery or had a myocardial infarction. In one aspect, the methodsinclude harvesting of a selected ECM tissue, freezing the selected ECMtissue, thawing the selected ECM tissue, and isolating an ECM material.The isolated ECM material is subjected to incubation and rinsing beforeit is processed in supercritical carbon dioxide and subsequently exposedto rapid depressurization. During or after the rapid depressurization ofthe ECM material, one or more additives can be incorporated into the ECMmaterial to impart desired characteristics to the resulting ECMcomposition. Rapid depressurization enhances the incorporation of theone or more additives into the ECM composition. Sterilized, acellularECM compositions produced using the disclosed methods are alsodisclosed.

Additional advantages of the disclosed methods and compositions will beset forth in part in the description which follows and, in part, will beunderstood from the description, or may be learned by practice of thedisclosed methods and compositions. The advantages of the disclosedmethods and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the preferred embodiments of the inventionwill become more apparent in the detailed description in which referenceis made to the appended drawings wherein:

FIGS. 1-2 show the results of an experiment in which DNA content wasmeasured for small intestinal submucosa (SIS) compositions followingvarious sterilization methods, including the sterilization methodsdescribed herein. FIG. 1 shows the DNA content of each SIS compositionfollowing sterilization. FIG. 2 shows the percentage of DNA that wasremoved from each SIS composition following sterilization, as comparedto raw, unprocessed SIS.

FIGS. 3-4 show the results of an experiment in which native growthfactor content was measured for SIS compositions following varioussterilization methods, including the sterilization methods describedherein. FIG. 3 shows the bFGF content of each SIS composition(normalized by dry weight of samples) following sterilization. FIG. 4shows the active TGF-β content of each SIS composition (normalized bydry weight of samples) following sterilization.

FIG. 5 shows the results of an experiment in which bFGF was incorporatedinto SIS compositions during rapid depressurization, as describedherein. FIG. 5 shows the bFGF content for each SIS composition(normalized by dry weight of samples) following rapid depressurization.

FIG. 6 shows the results of an experiment in which the tensile strengthof two-ply SIS compositions was measured following various sterilizationmethods, including the sterilization methods described herein. FIG. 6shows the tensile strength measured for each SIS composition followingsterilization.

FIG. 7 shows the results of an experiment in which native growth factorcontent was measured for SIS compositions following varioussterilization and/or decellularization methods, including thesterilization and decellularization methods described herein. FIG. 7shows the bFGF enzyme-linked immunosorbent assay (ELISA) results foreach SIS composition (normalized by dry weight of samples) followingsterilization and/or decellularization.

FIG. 8 shows the DNA content in SIS after it is processed in variousways. The baseline measurement is raw. The tissue was then exposed tosupercritical CO, followed by rapid depressurization (RDP) to facilitateenhanced removal of DNA and cellular debris. After the RDP, the tissuewas placed in supercritical CO₂ with peracetic acid (PAA) forsterilization. The comparison is to processed SIS either unsterilized orsterilized with ethylene oxide (ETO).

FIG. 9 shows the Percent removal of DNA from SIS after it is processedin various ways. The baseline measurement is raw. The tissue was thenexposed to supercritical CO₂ followed by rapid depressurization (RDP) tofacilitate enhanced removal of DNA and cellular debris. After the RDP,the tissue was placed in supercritical CO₂ with peracetic acid (PAA) forsterilization. The comparison is to processed SIS either unsterilized orsterilized with ethylene oxide (ETO).

FIG. 10 shows the variable active Transforming Growth Factor (TGF-beta)content in SIS after it is processed in various ways. The baselinemeasurement is raw, or unprocessed SIS followed by processing with onlyTriton X-100 (TX-100) detergent. The tissue was then exposed tosupercritical CO₂ followed by rapid depressurization (RDP) to facilitateenhanced removal of DNA and cellular debris. After the RDP, the tissuewas placed in supercritical CO? with peracetic acid (PAA) forsterilization. The comparison is to processed SIS either unsterilized orsterilized with ethylene oxide (ETO).

FIG. 11 shows the variable basic Fibroblast Growth Factor (bFGF) contentin SIS after it is processed in various ways. The baseline measurementis raw, or unprocessed SIS followed by processing with only Triton X-100(TX-100) detergent. The tissue was then exposed to supercritical CO₂followed by rapid depressurization (RDP) to facilitate enhanced removalof DNA and cellular debris. After the RDP, the tissue was placed insupercritical CO₂ with peracetic acid (PAA) for sterilization. Thecomparison is to processed SIS either unsterilized or sterilized withethylene oxide (ETO).

FIG. 12 shows the addition of basic Fibroblast Growth Factor (bFGF)content to SIS using rapid depressurization. The baseline measurement israw, or unprocessed SIS. The comparison is to processed SIS eitherunsterilized or sterilized with ethylene oxide (ETO).

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to thefollowing detailed description, examples, and claims, and their previousand following description. However, before the present devices, systems,and/or methods are disclosed and described, it is to be understood thatthis invention is not limited to the specific devices, systems, and/ormethods disclosed unless otherwise specified, as such can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only and is notintended to be limiting.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a peptide is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the peptide are discussed, each and every combination andpermutation of peptide and the modifications that are possible arespecifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as aclass of molecules D, E, and F and an example of a combination molecule,A-D is disclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, in this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C—F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed, it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the appended claims.

It is understood that the disclosed methods and compositions are notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed methods and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present methods andcompositions, the particularly useful methods, devices, and materialsare as described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of such compounds; reference to “thecompound” is a reference to one or more compounds and equivalentsthereof known to those skilled in the art, and so forth.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed, then “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data are provided in a number of differentformats and that these data represent endpoints, starting points, andranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units is also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to” and is not intended toexclude, for example, other additives, components, integers or steps.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon. Nothing herein is tobe construed as an admission that the present invention is not entitledto antedate such disclosures by virtue of prior invention. No admissionis made that any reference constitutes prior art. The discussion ofreferences states what their authors assert, and applicants reserve theright to challenge the accuracy and pertinence of the cited documents.

A. METHODS

Disclosed herein are methods of treating or preventing a cardiacarrhythmia in a subject. The methods can comprise administering to thecardiac tissue of the subject a therapeutically effective amount of acomposition comprising a mammalian extracellular matrix (ECM).

In some aspects, the mammalian ECM is derived from a native source. Insome aspects, the mammalian ECM is produced in vitro using mammaliancells. In some aspects, the mammalian ECM is extracted directly frommammalian tissue/organs. In some aspects the composition comprisingmammalian ECM further comprises synthetic ECM.

In some aspects, the composition comprising a mammalian ECM inhibitsscar formation. In some aspects, the composition comprising a mammalianECM promotes regeneration of damaged tissue. In some aspects, thecomposition comprising a mammalian ECM inhibits inflammation.

By “treatment” is meant the medical management of a patient with theintent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

By “prevent” or “preventing” is meant reducing the frequency or severityof a disease or condition. The term does not require an absolutepreclusion of the disease or condition. Rather, this term includesdecreasing the chance for disease occurrence. Thus, disclosed aremethods of reducing the occurrence and/or severity of a cardiacarrhythmia in a subject, comprising administering to cardiac tissue ofthe subject a therapeutically effective amount of a compositioncomprising a mammalian ECM.

The term “therapeutically effective” means that the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses, symptoms, or sequelae of a disease or disorder. Suchamelioration only requires a reduction or alteration, not necessarilyelimination, of the causes, symptoms, or sequelae of a disease ordisorder.

As used herein, the term “cardiac tissue” includes the myocardium,epicardium, endocardium, and pericardium (the pericardial sac) of theheart. The term as used herein also refers to the great vessels leadingto or from the heart. The term as used herein also refers to portions ofthe vagus nerve that innervate the heart.

Thus, in some aspects, the methods comprise administering a compositioncomprising a mammalian ECM to the heart of the subject. In some aspects,the methods comprise administering a composition comprising a mammalianECM to the myocardium of the subject. The myocardium can be ventricularmyocardium. The myocardium can be atrial myocardium. In some aspects,the methods comprise administering a composition comprising a mammalianECM to the epicardium of the subject. In some aspects, the methodscomprise administering a composition comprising a mammalian ECM to theendocardium of the subject. In some aspects, the methods compriseadministering a composition comprising a mammalian ECM to thepericardium of the subject. In some aspects, the methods compriseadministering a composition comprising a mammalian ECM into the spacebetween the epicardium and the pericardium of the subject.

In some aspects, the methods comprise administering a compositioncomprising a mammalian ECM to a great vessel of the subject. In someaspects, the vessel is the superior vena cava, inferior vena cava,pulmonary vein, pulmonary artery, or aorta of the subject. For example,the method can comprise administering a composition comprising amammalian ECM to the adventitia (external portion) of one or more of thegreat vessels. In some aspects, the method comprises administering acomposition comprising a mammalian ECM to the cardiac circulation. Thus,the method comprises administering a composition comprising a mammalianECM into a blood vessel or heart chamber.

Parasympathetic innervation of the heart is mediated by the vagus nerve.The right vagus innervates the sinoatrial (SA) node. Parasympathetichyperstimulation predisposes those affected to bradyarrhythmias. Theleft vagus when hyperstimulated predisposes the heart toatrioventricular (AV) blocks. Thus, in some aspects, the methodscomprise administering a composition comprising a mammalian ECM to aportion of the vagus nerve of the subject that innervates the heart.

As used herein, the term “subject” means any individual who is thetarget of administration. The subject can be a vertebrate, for example,a mammal. Thus, the subject can be a human. The term does not denote aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are intended to be covered. A patientrefers to a subject afflicted with a disease or disorder. The term“patient” includes human and veterinary subjects. As used herein, theterms “patient” and “subject” can be used interchangeably.

In some aspects, the subject of the disclosed method has been identifiedas being at risk of developing a cardiac arrhythmia. In some aspects,the subject of the disclosed method has undergone heart surgery,including, but not limited to, open-heart surgery. In some aspects, thesubject of the disclosed method has undergone multiple combined heartprocedures, including, but not limited to, open heart procedures. Insome aspects, the subject of the disclosed method has undergone heartvalve surgery. In some aspects, the subject of the disclosed method isat least 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85years of age. In some aspects, the composition is administered to asubject who has had a myocardial infarction. In some aspects, thesubject of the disclosed method has emphysema or chronic obstructivepulmonary disease. In some aspects, the subject of the disclosed methodhas a history of arrhythmia.

In some aspects, the disclosed method does not comprise administering apatch comprising small intestinal submucosa (SIS) to an opening in thepericardial sac of the heart. In some aspects, the disclosed method doesnot consist of administering a patch comprising small intestinalsubmucosa (SIS) to an opening in the pericardial sac of the heart. Insome aspects, the mammalian ECM is not SIS. Thus, in some aspects, thecomposition comprising mammalian ECM does not consist of SIS. In someaspects, the composition comprising mammalian ECM is not a patch. Insome aspects, the disclosed method does not comprise administering thecomposition comprising mammalian ECM as a patch to an opening in thepericardial sac of the heart. In some aspects, the cardiac tissue of thedisclosed method is not pericardium. In some aspects, the disclosedmethod does not comprise administering the composition to thepericardium.

In other aspects, however, the disclosed method comprises administeringa patch comprising small intestinal submucosa (SIS) to an opening in thepericardial sac of the heart. The compositions used in the disclosedmethods can comprise one or more additional agents (i.e., additives)such as growth factors, cytokines, proteoglycans, glycosaminoglycans(GAGs), proteins, peptides, nucleic acids, small molecules, cells andpharmaceutical agents, such as statin drugs, corticosterioids,anti-arrhythmic drugs, nonsteroidal anti-inflammatory drugs, otheranti-inflammatory compounds, nanoparticles, and metallic compounds. Inother aspects, the disclosed method comprises administering a patchcomprising small intestinal submucosa (SIS) to an opening in thepericardial sac of the heart, but the method further comprisesadditional steps.

Also disclosed herein is a method of treating or preventing a cardiacarrhythmia in a subject, comprising administering to cardiac tissue ofthe subject a therapeutically effective amount of a compositioncomprising a mammalian extracellular matrix and further comprising ananti-arrhythmic drug, a lipid-lowering drug, cells, a protein, or acombination thereof

1. Cardiac Arrhythmia

Cardiac arrhythmia (also referred to as dysrhythmia) is a term for anyof a large and heterogeneous group of conditions in which there isabnormal electrical activity in the heart. The heart beat (pulse) can betoo fast or too slow and can be regular or irregular.

Some arrhythmias are life-threatening medical emergencies that canresult in cardiac arrest and sudden death. Others cause symptoms such asan abnormal awareness of heart beat (palpitations) and can be merelyannoying. Others may not be associated with any symptoms at all butpredispose toward potentially life-threatening stroke or embolus.

The term sinus arrhythmia refers to a normal phenomenon of mildacceleration and slowing of the heart rate that occurs with breathing inand out. It is usually quite pronounced in children and steadily lessenswith age. This can also present during meditation breathing exercisesthat involve deep inhaling and breath-holding patterns.

Each heart beat originates as an electrical impulse from a small area oftissue in the right atrium of the heart called the sinus node orsinoatrial (SA) node. The impulse initially causes both atria tocontract and then activates the atrioventricular (or AV) node, which isnormally the only electrical connection between the atria and theventricles, or main pumping chambers. The impulse then spreads throughboth ventricles via the His Purkinje fibers causing a synchronizedcontraction of the ventricular myocardium.

A heart rate less than 60 beats per minute is a bradycardia. This can becaused by a slowed signal from the sinus node (termed sinusbradycardia), a pause in the normal activity of the sinus node (termedsinus arrest), or by blocking of the electrical impulse on its way fromthe atria to the ventricles (termed AV block or heart block). Heartblock comes in varying degrees and severity. It can be caused byreversible poisoning of the AV node (with drugs that impair conduction)or by irreversible damage to the node.

A heart rate faster than 100 beats per minute is a tachycardia.Tachycardia can result in palpitation; however, tachycardia is notnecessarily an arrhythmia. Increased heart rate is a normal response tophysical exercise or emotional stress. This is mediated by thesympathetic nervous system's effect on the sinus node and is calledsinus tachycardia. Other things that increase sympathetic nervous systemactivity in the heart include ingested or injected substances such ascaffeine or amphetamines, and an overactive thyroid gland(hyperthyroidism). Heart rate can be increased with sympathomimeticdrugs.

Tachycardia that is not sinus tachycardia usually results from theaddition of abnormal impulses that can begin by one of three mechanisms:automaticity, re-entry or triggered activity.

Automaticity refers to a cardiac muscle cell firing off an impulse onits own. All of the cells in the heart have the ability to initiate anaction potential; however, only some of these cells are designed toroutinely trigger heart beats. These cells are found in the conductionsystem of the heart and include the SA node, AV node, Bundle of His andPurkinje fibers. The SA node is a single specialized location in theatrium which has a higher automaticity (a faster pacemaker) than therest of the heart and therefore is usually responsible for setting theheart rate and initiating each heart beat. Any part of the heart thatinitiates an impulse without waiting for the SA node is called anectopic focus and is by definition a pathological phenomenon. This cancause a single premature beat now and then, or, if the ectopic focusfires more often than the SA node, it can produce a sustained abnormalrhythm. Conditions that increase automaticity include sympatheticnervous system stimulation and hypoxia. The resulting heart rhythmdepends on where the first signal begins. If it is the SA node, therhythm remains normal but rapid; if it is an ectopic focus, many typesof arrhythmia can result.

Re-entry arrhythmias occur when an electrical impulse recurrentlytravels in a tight circle within the heart, rather than moving from oneend of the heart to the other and then stopping. Every cardiac cell isable to transmit impulses in every direction but can only do so oncewithin a short period of time. Normally, the action potential impulsewill spread through the heart quickly enough that each cell will onlyrespond once. However, if conduction is abnormally slow in some areas,part of the impulse will arrive late and potentially be treated as a newimpulse. Depending on the timing, this can produce a sustained abnormalcircuit rhythm. Re-entry circuits are responsible for atrial flutter,most paroxysmal supraventricular tachycardias, and dangerous ventriculartachycardia. When an entire chamber of the heart is involved in multiplemicro-reentry circuits and therefore quivering with chaotic electricalimpulses, it is said to be in fibrillation.

Fibrillation can affect one or both atria (atrial fibrillation) or oneor both ventricles (ventricular fibrillation). If left untreated,ventricular fibrillation (VF, or V-fib) can lead to death withinminutes.

Triggered beats occur when problems at the level of the ion channels inindividual heart cells result in abnormal propagation of electricalactivity and can lead to sustained abnormal rhythm. Triggered beats arerelatively rare but can result from the action of anti-arrhythmic drugs.

Arrhythmia can be classified by rate (physiological, tachycardia,bradycardia), or mechanism (automaticity, re-entry, fibrillation).

It is also appropriate to classify arrhythmia by site of origin. Forexample, atrial arrhythmias include premature atrial contractions(PACs), wandering atrial pacemaker, multifocal atrial tachycardia,atrial flutter, and atrial fibrillation (Afib). Junctional arrhythmiasinclude supraventricular tachycardia (SVT), AV nodal re-entranttachycardia (the most common cause of paroxysmal supraventriculartachycardia (PSVT)), junctional rhythm, junctional tachycardia, andpremature junctional complex. Atrioventricular arrhythmias include AVre-entrant tachycardia (occurs when a re-entry circuit crosses betweenthe atria and ventricles somewhere other than the AV node).

Ventricular arrhythmias include premature ventricular contractions (PVC)(sometimes called ventricular extra beats (VEBs)), acceleratedidioventricular rhythm, monomorphic ventricular tachycardia, polymorphicventricular tachycardia, and ventricular fibrillation.

Heart blocks (also known as AV blocks, the most common causes ofbradycardia) include first degree heart block (PR interval greater than200 msec in length on the surface ECG), second degree heart block (Types1 and 2), and third degree heart block (also known as complete heartblock).

Cardiac arrhythmias are often first detected by auscultation of theheartbeat with a stethoscope or by feeling peripheral pulses. Thesemethods cannot usually diagnose specific arrhythmias but can give ageneral indication of the heart rate and whether it is regular orirregular. Not all of the electrical impulses of the heart produceaudible or palpable beats; in many cardiac arrhythmias, the premature orabnormal beats do not produce an effective pumping action and areexperienced as “skipped” beats.

The simplest specific diagnostic test for assessment of heart rhythm isthe electrocardiogram (abbreviated ECG or EKG). A Holter monitor is anEKG recorded over a 24-hour period, to detect arrhythmias that canhappen briefly and unpredictably throughout the day.

Sudden arrhythmia death syndrome (SADS) is a term used to describesudden death due to cardiac arrest brought on by an arrhythmia. Often,the subject has no symptoms before dying suddenly. The most common causeof sudden death in the United States is coronary artery disease.Approximately 300,000 people die suddenly of this cause every year inthe United States. SADS can also be caused by, for example, manyinherited conditions and heart diseases that can affect young people.

In children, for example, viral myocarditis, long Q-T syndrome, Brugadasyndrome, Catecholaminergic polymorphic ventricular tachycardia andhypertrophic cardiomyopathy, and arrhythmogenic right ventriculardysplasia can cause SADS.

In some aspects, a cardiac arrhythmia is atrial fibrillation orventricular fibrillation.

1. Administration

In some aspects, the mammalian ECM is a patch in a form such as a sheet,plug, a laminate, a weave, a polymer matrix, a plurality of strands, asponge, or one or more strips. As used herein, a “sponge” can be aresilient, absorbent, porous composition comprising fibers of ECM. Inone aspect, the fibers can be interlacing. A sponge can be used todeliver one or more of the disclosed additional agents (i.e., additives)to heart tissue. Thus, in some aspects, the mammalian ECM is placed intodirect contact with the cardiac tissue of a subject during heartsurgery. In some aspects, the composition comprising a mammalian ECM isadministered to an opening in the pericardial sac of the heart. In someaspects, the composition overlaps the opening in the pericardial sac.Thus, the composition comprising a mammalian ECM can be administered tothe surgical opening of the pericardium during or after heart surgery.In another aspect, the mammalian ECM can be placed into contact withcardiac structures, such as the great vessels, e.g., aorta, pulmonaryartery, pulmonary vein, superior vena cava, and inferior vena cava. Insome aspects, the mammalian ECM composition, for example a sponge, canbe sandwiched between and in contact with the epicardium and the innerwall of the pericardial sac.

Wherein the mammalian ECM is in a solid form such as a sheet, a plug, alaminate, a weave, a polymer matrix, a plurality of strands, a sponge,or one or more strips, the composition can be attached to the cardiactissue using standard means available in the art. For example, thecomposition comprising mammalian ECM can be attached to the cardiactissue with sutures, bioadhesives such as fibrin glue, staples, and thelike.

The disclosed compounds and compositions comprising a mammalian ECM canbe administered in any suitable manner. For example, the compositionscan be administered parenterally (e.g., intramuscular injection),topically or the like. Thus, in some aspects, the composition comprisinga mammalian ECM is injectable. The disclosed compositions can beinjected into the cardiac tissue using ordinary means. For example, thecomposition comprising a mammalian ECM can be delivered to the cardiactissue via a syringe or a cardiac or coronary catheter. Cardiaccatheterization (heart cath) is the insertion of a catheter into achamber or vessel of the heart. This can be done for both diagnosticand/or interventional purposes. Coronary catheterization is a subset ofthis technique, involving the catheterization of the coronary arteries.

Thus, in some aspects, the composition comprising a mammalian ECM can beinjected into the myocardium of the heart. In some aspects, thecomposition comprising a mammalian ECM can be injected into theepicardium of the heart. In some aspects, the composition comprising amammalian ECM can be injected into the endocardium of the heart. In someaspects, the composition comprising a mammalian ECM can be injected intothe pericardium of the heart. In some aspects, the compositioncomprising a mammalian ECM can be injected between layers of the heart,e.g., between the pericardium and epicardium, between the epicardium andmyocardium, and between the myocardium and endocardium.

In some aspects, the composition comprising a mammalian ECM can beadministered to the atrial or ventricular septum of the subject. Forexample, in some aspects, the composition comprising a mammalian ECM canbe administered to a ventricular septal defect. A ventricular septaldefect (VSD) is a defect in the ventricular septum, the wall dividingthe left and right ventricles of the heart. The ventricular septumconsists of an inferior muscular and superior membranous portion and isextensively innervated with conducting cardiomyocytes. The membranousportion, which is close to the atrioventricular node, is most commonlyaffected in adults and older children. Congenital VSDs are collectivelythe most common congenital heart defects.

In some aspects, the composition comprising a mammalian ECM can beadministered to an atrial septal defect (ASD). An ASD is a form ofcongenital heart defect that enables blood flow between the left andright atria via the interatrial septum. The interatrial septum is thetissue that divides the right and left atria. Without this septum, or ifthere is a defect in this septum, it is possible for blood to travelfrom the left side of the heart to the right side of the heart, or viceversa. Irrespective of interatrial communication bi-directions, thisresults in the mixing of arterial and venous blood. The mixing ofarterial and venous blood may or may not be hemodynamically significant,if even clinically significant. This mixture of blood may or may notresult in what is known as a “shunt.” The amount of shunting present, ifany, dictates hemodynamic significance (see Pathophysiology below). A“right-to-left-shunt” typically poses the more dangerous scenario (seePathophysiology below).

The mammalian ECM can be in an aerosol form. Thus, in some aspects, themammalian ECM can be sprayed on the cardiac tissue of the subject.

The mammalian ECM can be in a particulate form. Particulate mammalianECM can be administered by injecting an emulsified composition,spraying, layering, packing, dusting, painting, or other similar typesof application of the dry particulate, the liquid composition, or thesemi-solid compositions.

In some aspects, the composition is administered to the epicardialsurface of the heart. Thus, in some aspects, the composition isinjected, sprayed, or attached to the epicardial surface of the heart.

The exact amount of the compositions required can vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, and the severity of the disorder being treated. Thus, it isnot possible to specify an exact amount for every composition. However,an appropriate amount can be determined by one of ordinary skill in theart using only routine experimentation given the teachings herein. Thus,effective dosages and schedules for administering the compositions canbe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptom or disorder is affected. The dosage should not be solarge as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage can vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen and can be determined by one of skill in theart. Dosage can vary and can be administered in one or more doseadministrations daily, for one or several days. Guidance can be found inthe literature for appropriate dosages for given classes ofpharmaceutical products.

Following administration of a disclosed composition for treating,inhibiting, or preventing a cardiac arrhythmia, the efficacy of themethod can be assessed in various ways well known to the skilledpractitioner. For example, one of ordinary skill in the art willunderstand that a composition disclosed herein is efficacious intreating a cardiac arrhythmia in a subject using an electrocardiogram.

The compositions disclosed herein can be administered prophylacticallyto subjects who are at risk for cardiac arrhythmia. The disclosedcompositions and methods can also be used, for example, as tools toisolate and test new drug candidates for treating or preventing cardiacarrhythmia. The disclosed compositions can also be used in a variety ofways as research tools. Other uses are disclosed, apparent from thedisclosure, and/or will be understood by those in the art.

1. Combination Therapy

The herein disclosed methods can further comprise treating the subjectwith conventional anti-arrhythmia therapies. For example, there are manyclasses of anti-arrhythmic medications with different mechanisms ofaction and many different individual drugs within these classes. Thus,the method can further comprise administering to the subject one or moreanti-arrhythmic medications.

Some arrhythmias, e.g., atrial fibrillation, cause blood clotting withinthe heart and increase risk of embolus and stroke. Anticoagulantmedications such as warfarin and heparin, and anti-platelet drugs suchas aspirin can reduce the risk of clotting. Thus, the method can furthercomprise administering to the subject an anticoagulant.

Arrhythmias can also be treated electrically, by applying a shock acrossthe heart, either externally to the chest wall or internally to theheart via implanted electrodes or intra-operatively. Cardioversion canbe achieved either pharmacologically or via the application of a shocksynchronized to the underlying heartbeat. It is used for treatment ofsupraventricular tachycardias. In elective cardioversion, the recipientis usually sedated or lightly anesthetized for the procedure. Forexample, atrial flutter can be treated by cardioversion. Thus, themethod can further comprise treating the subject with cardioversion.

With synchronized cardioversion, a reversion shock is delivered by wayof pads or paddles of a selected amount of electric current over apre-defined number of milliseconds at the optimal moment in the cardiaccycle which corresponds to the R wave of the QRS complex on the ECGTiming the shock to the R wave prevents the delivery of the shock duringthe vulnerable period (or relative refractory period) of the cardiaccycle, which could induce ventricular fibrillation.

Defibrillation differs from cardioversion in that the shock is notsynchronized to a cardiac cycle. It is needed for the chaotic rhythm ofventricular fibrillation and is also used for pulseless ventriculartachycardia. Often, more electricity is required for defibrillation thanfor cardioversion. Because most subjects with ventricular fibrillationare unconscious, there is generally no need for sedation. Thus, themethod can further comprise treating the subject with defibrillation.

Defibrillation or cardioversion can be accomplished by an implantablecardioverter-defibrillator (ICD). Thus, the method can further compriseadministering to the subject an ICD.

Electrical treatment of arrhythmia also includes cardiac pacing.Temporary pacing can be necessary for reversible causes of very slowheartbeats, or bradycardia, (for example, from drug overdose ormyocardial infarction). A permanent pacemaker can be placed insituations where the bradycardia is not expected to recover. Thus, themethod can further comprise administering to the subject a pacemaker.

Fine probes can in some aspects be inserted through the blood vessels tomap electrical activity from within the heart. This allows abnormalareas of conduction to be located very accurately, and subsequentlydestroyed with heat, cold, electrical or laser probes.

A. COMPOSITIONS

A patch of mammalian ECM has been shown to act as a mechanical scaffoldwhile the body recruits the necessary cells to remodel and repair thecardiac tissue. Disclosed herein is the surprising ability of mammalianECM to additionally treat and/or prevent cardiac arrhythmia. Thus,disclosed herein are compositions comprising mammalian ECM for use inthe disclosed method(s) for treating or preventing cardiac arrhythmia ina subject. The disclosed compositions can be natural or synthetic. Thecompositions can be de-cellularized or comprise cells such as stemcells.

The herein disclosed compositions comprising mammalian ECM can be in theform of, for example, a patch, an emulsion, an injectable solution, agel, a fluid, a paste, a powder, a strand, a sponge, a strip, a spray, avapor, an aerosol, a cream, or a coating. The composition can furthercomprise one or more additional components, including, for example, acell, peptide, polypeptide, protein or other biological moieties. Wherethe composition is a patch, it can be in a form selected from a sheet, alaminate, a weave, a polymer matrix, a plurality of strands, a sponge,one or more strips, or a combination thereof.

The herein disclosed compositions comprising mammalian ECM can be madeinto a particulate and fluidized as described in U.S. Pat. No. 5,275,826to Badylak, U.S. Pat. No. 6,579,538 to Spievack, and U.S. Pat. No.6,933,326 to Griffey. Fluidized or emulsified compositions (the liquidor semi-solid forms) can be present at a certain concentration, forexample at a concentration of extracellular matrix greater than about0.001 mg/ml. The concentration of these liquid or semi-solid componentsof the extracellular matrix composition can be in a range from about0.001 mg/ml to about 200 mg/ml. The concentrations can further be foundin more specific ranges such as for example the following set of ranges:about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 125 mg/ml,about 25 mg/ml to about 100 mg/ml, about 20 mg/ml to about 75 mg/ml,about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 50 mg/ml, andabout 35 mg/ml to about 45 mg/ml, and about 40 mg/ml. to about 42 mg/ml.This set of ranges is exemplary and not intended to be exhaustive. It iscontemplated that any value within any of these specifically listedranges is a reasonable and useful value for a concentration of a liquid,emulsion, gel, paste or other liquid or semi-solid component of thecomposition.

1. Mammalian Extracellular Matrix

Extracellular matrix materials act as a natural scaffold for repairingsoft tissues in the body. Animal studies have shown that the originalextracellular matrix material remodels and is replaced by host tissue.Mammalian ECM is a resorbable biomaterial which has been usedsuccessfully as a xenogenic tissue graft that induces constructiveremodeling of a variety of animal tissues including blood vessels,urinary bladder, dura, abdominal wall, tendons and ligaments. Examplesof mammalian ECM include small intestine submucosa (SIS), urinarybladder submucosa (UBS), stomach submucosa (SS), or liver basementmembrane (LBM).

The remodeling process includes rapid neovascularization and abundantaccumulation of mesenchymal and epithelial cells that support extensivedeposition of a new extracellular matrix. The noncollagenous portion of,for example, the SIS extracellular matrix is composed of variousglycoproteins, such as hyaluronic acid, heparin, dermatan andchondroitin sulfate A, as well as FGF-2 and TGF-β growth factors.

After processing, mammalian ECM can retain many of the endogenousproteins, which act as growth and differentiation factors. These factorsstimulate the local environment to populate the mammalian ECM with cellsthat are then able to differentiate into the original tissue that themammalian ECM is replacing.

Mammalian ECM is a scaffold matrix of polymerized “structural” proteinsthat fit into three groups: collagens, glycoproteins, and proteoglycans(which have glycosaminoglycan repeats throughout). These moleculesactually polymerize to form the scaffold or matrix of proteins thatexists in dynamic interaction with cells and closely placed functionalproteins (either on the cells, or bound to a structural protein). Thus,mammalian ECM also includes within its matrix scaffold “functional”proteins that interact with the structural proteins and with migratingor recruited cells, such as stem cells. The matrix functional proteinsalso interact with protein-expressing cells during the life andmaintenance of the matrix scaffold itself as it rebuilds and maintainsits components. Some proteins can be both a structural and functionalprotein, depending on the protein's configuration and placement in thewhole matrix.

The ECM of, for example, cardiac tissue is made up of collagen types I(predominant), III, IV, V, and VI, combined which are 92% of the dryweight of the matrix. The ECM of cardiac tissue is also made up ofglycosaminoglycans (GAGs), which include chondroitin sulfate A and B,heparan, heparin, and hyaluronic acid. Glycoproteins such as fibronectinand entactin, proteoglycans such as decorin and perlecan, and growthfactors such as transforming growth factor beta (TGF-β), fibroblastgrowth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF)are key players in the activity of a myocardium regenerating matrix.Furthermore, the precise chemical constitution of the matrix appears toplay a role in its function, including, for example, what collagen typeis prevalent in the matrix. Thus, the outcome of any tissue regenerativeprocesses can be determined by the structural and functional componentsof the matrix scaffold that form the basis of the regenerative process.

Facilitating cell adhesion functions in ECM are cell adhesion molecules(CAMs). The CAMs can either be available endogenously or added as anadditional component of the composition. CAMs are glycoproteins lodgedin the surface of the cell membrane or transmembrane connected tocytoskeletal components of the cell. Specific CAMs include cadherinsthat are calcium dependent, and more than 30 types are known. Alsoworking as CAMs are integrins which are proteins that link thecytoskeleton of the cell in which they are lodged to the extracellularmatrix or to other cells through alpha and beta transmembrane subunitson the integrin protein. Cell migration, embryogenesis, hemostasis, andwound healing are facilitated by the integrins in the matrix. Syndecansare proteoglycans that combine with ligands for initiating cell motilityand differentiation. Immunoglobulins provide any necessary immune andinflammatory responses. Selectins promote cell-cell interactions.

i. Native Sources and Preparations

In some aspects, the mammalian ECM is derived from native source. Nativeextracellular matrix scaffolds and the proteins that form them can befound in their natural environment, i.e., the extracellular matrices ofmammals. These materials can be prepared for use in mammals in tissuegraft procedures.

In some aspects, the mammalian ECM is extracted from mammaliantissue/organs. For example, in some aspects, the mammalian ECM comprisesthe basement membrane (or transitional epithelial layer), tunicapropria, tunica submucosa, tunica muscularis, tunica serosa, or acombination thereof from a mammalian tissue source. Thus, in someaspects, the mammalian ECM comprises the basement membrane (ortransitional epithelial layer) from a mammalian tissue source. In someaspects, the mammalian ECM comprises the subjacent tunica propria from amammalian tissue source. In some aspects, the mammalian ECM comprisesthe tunica submucosa from a mammalian tissue source. In some aspects,the mammalian ECM comprises the tunica muscularis from a mammaliantissue source. In some aspects, the mammalian ECM comprises the tunicaserosa from a mammalian tissue source.

For example, small intestine submucosa (SIS) is described in U.S. Pat.No. 5,275,826; urinary bladder submucosa (UBS) is described in U.S. Pat.No. 5,554,389; stomach submucosa (SS) is described in U.S. Pat. No.6,099,567; and liver basement membrane (LBM) is described in U.S. Pat.No. 6,379,710, each of which is incorporated herein by reference forteachings of how to make and use these native extracellular matrices.

Thus, in some aspects, the mammalian ECM of the disclosed compositionsand methods is small intestine submucosa (SIS). In some aspects, themammalian ECM of the disclosed compositions and methods is urinarybladder submucosa (UBS). In some aspects, the mammalian ECM of thedisclosed compositions and methods is stomach submucosa (SS). In someaspects, the mammalian ECM of the disclosed compositions and methods isliver basement membrane (LBM).

In some aspects, the mammalian ECM of the disclosed compositions andmethods is from dermis. For example, AlloDerm®, produced by LifeCellCorporation, is an acellular tissue matrix which is produced from normalhuman skin using processing techniques established to remove theepidermis and cells within the dermis without significantly altering thenormal biochemistry and molecular architecture of the connective tissuematrix. The resulting product is in a freeze-dried form allowingextended shelf-life and ease of shipping without degradation or loss ofthe normal tissue matrix components. AlloDerm® can retain decorin,hyaluronic acid, chondroitin sulfates, nidogen, growth factors and otherbiochemical proteins present in normal soft tissues. Additionally,AlloDerm® can contain the basement membranes of vascular channels andthe orientation of elastin and collagen fibers of the starting dermaltissue.

In some aspects, the mammalian ECM of the disclosed compositions andmethods is from fascia. In some aspects, the mammalian ECM of thedisclosed compositions and methods is from parenchymal tissue. In someaspects, the mammalian ECM of the disclosed compositions and methods isfrom pericardium. In some aspects, the mammalian ECM of the disclosedcompositions and methods is myocardial extracellular matrix. In someaspects, the mammalian ECM of the disclosed compositions and methods isfrom decellularized heart tissue, produced, for example, by coronaryartery perfusion with detergents (Ott, H C, et al. Nat. Med. 2008February; 14(2):213-21).

In some aspects, the mammalian ECM comprises a collagen scaffold derivedfrom a mammalian tissue or organ source. The collagen scaffold frommammalian source can in some aspects comprise the basement membrane ofthe mammalian tissue source.

In some aspects, the mammalian ECM is produced in vitro. For example,the mammalian ECM can be produced from culture of mammalian cells. Themammalian ECM can be produced from proteins extracted from mammaliantissue/organs. For example, in some aspects, the mammalian ECM comprisesan artificial collagen scaffold synthesized from collagen extracted froma mammalian tissue or organ source. Collagen from mammalian sources canbe retrieved from matrix-containing tissues and used to form a matrixcomposition. Extracellular matrices can be synthesized from cellcultures as in the product manufactured by Matrigel™. In addition,dermal extracellular matrix material, subcutaneous extracellular matrixmaterial, large intestine extracellular matrix material, placentalextracellular matrix material, omentum extracellular matrix material,heart extracellular matrix material, and lung extracellular matrixmaterial, can be used, derived and preserved similarly as describedherein for the SIS, SS, LBM, and UBS materials. Other organ tissuesources of basement membrane for use in accordance with the disclosedcompositions and methods include, but are not limited to, spleen, lymphnodes, salivary glands, prostate, pancreas and other secreting glands.In general, any tissue of a mammal that has an extracellular matrix canbe used for developing an extracellular matrix component.

Collagenous matrix can be selected from a variety of commerciallyavailable collagen matrices or can be prepared from a wide variety ofnatural sources of collagen. Collagenous matrix for use in accordancewith the disclosed compositions and methods can comprise highlyconserved collagens, glycoproteins, proteoglycans, andglycosaminoglycans in their natural configuration and naturalconcentration. Collagens can be from animal sources, from plant sources,or from synthetic sources, all of which are available and standard inthe art.

The proportion of scaffold material in the composition when nativescaffold is used can be large, as the natural balance of extracellularmatrix proteins in the native scaffolds usually represents greater than90% of the extracellular matrix material by dry weight. Thus, thescaffold component of the composition by weight can be generally greaterthan 50% of the total dry weight of the composition. The scaffold cancomprise an amount of the composition by weight greater than 60%,greater than 70%, greater than 80%, greater than 82%, greater than 84%,greater than 86%, greater than 88%, greater than 90%, greater than 92%,greater than 94%, greater than 96%, and greater than 98% of the totalcomposition.

Native extracellular matrices can be prepared with care that theirbioactivity for treating or preventing cardiac arrhythmia is preservedto the greatest extent possible. Key functions that can be preservedinclude control or initiation of cell adhesion, cell migration, celldifferentiation, cell proliferation, cell death (apoptosis), stimulationof angiogenesis, proteolytic activity, enzymatic activity, cellmotility, protein and cell modulation, activation of transcriptionalevents, provision for translation events, inhibition of somebioactivities, for example inhibition of coagulation, stem cellattraction, and chemotaxis. Assays for determining these activities arestandard in the art. For example, material analysis can be used toidentify the molecules present in the material composition. Also, invitro cell adhesion tests can be conducted to make sure that the fabricor composition is capable of cell adhesion.

The disclosed compositions comprising mammalian ECM can bedecellularized in order to render them non-immunogenic. In some aspects,the decellularization process is completed with some of the key proteinfunctions retained, either by replacement of proteins incidentallyextracted with the cells, or by adding exogenous cells to the matrixcomposition after cell extraction, which cells produce or carry proteinsinvolved in treating or preventing cardiac arrhythmia.

When adding proteins to the extracellular matrix composition, theproteins can be simply added with the composition, or each protein canbe covalently linked to a molecule in the matrix. Standardprotein-molecule linking procedures can be used to accomplish thecovalent attachment.

For decellularization when starting with a source tissue/organ as asource of mammalian ECM, source tissue/organ perfusion process can beused. The source tissue/organ can be perfused with a decellularizationagent, for example 0. 1% peracetic acid, rendering the organ acellular.The source tissue/organ can then be cut into portions and stored (e.g.,in aqueous environment, liquid nitrogen, cold, freeze-dried, orvacuum-pressed) for later use. Any appropriate decellularizing agent canbe used in source tissue/organ perfusion process. Further, disclosedbelow is a method of sterilizing and simultaneously decellularizing morecompletely an ECM material for use in the disclosed methods for treatingor preventing cardiac arrhythmia in a subject who has undergone heartsurgery or had a myocardial infarction.

With regard to submucosal tissue, extractions can be carried out nearneutral pH (in a range from about pH 5.5 to about pH 7.5) in order topreserve the presence of growth factors in the matrices. Alternatively,acidic conditions (i.e., less than pH 5.5) can be used to preserve thepresence of glycosaminoglycan components, at a temperature in a rangebetween 0 and 50 degrees centigrade. In order to regulate the acidic orbasic environment for these aqueous extractions, a buffer and chaotropicagent (generally at a concentration from about 2M to about 8M) can beselected, such as urea (at a concentration from about 2M to 4M),guanidine (at a concentration from about 2M to about 6M, most typicallyabout 4M), sodium chloride, magnesium chloride, and non-ionic or ionicsurfactants. Urea at 2M in pH 7.4 provides extraction of FGF-2 and theglycoprotein fibronectin. Using 4M guanidine with pH 7.4 buffer yields afraction having transforming growth factor beta. (TGF-β).

Because of the collagenous structure of basement membrane and the desireto minimize degradation of the membrane structure during celldissociation, collagen specific enzyme activity can be minimized in theenzyme solutions used in the cell-dissociation step. For example, sourcetissue/organ can be treated with a calcium chelating agent or chaotropicagent, such as a mild detergent, e.g., as Triton 100. The celldissociation step can also be conducted using a calcium chelating agentor chaotropic agent in the absence of an enzymatic treatment of thetissue/organ. The cell-dissociation step can be carried out bysuspending source tissue slices in an agitated solution containing about0.05 to about 2%, more typically about 0.1 to about 1% by weightprotease, optionally containing a chaotropic agent or a calciumchelating agent in an amount effective to optimize release andseparation of cells from the basement membrane without substantialdegradation of the membrane matrix.

After contacting the source tissue/organ with the cell-dissociationsolution for a time sufficient to release all cells from the matrix, theresulting tissue/organ basement membrane can be rinsed one or more timeswith saline and optionally stored in a frozen hydrated state or apartially dehydrated state until used as described below. Thecell-dissociation step can require several treatments with thecell-dissociation solution to release substantially all cells from thebasement membrane. The source tissue/organ can be treated with aprotease solution to remove the component cells, and the resultingextracellular matrix material is further treated to remove or inhibitany residual enzyme activity. For example, the resulting basementmembrane can be heated or treated with one or more protease inhibitors.

Basement membrane or other native extracellular matrix scaffolds can besterilized using conventional sterilization techniques including tanningwith glutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxidetreatment, propylene oxide treatment, gas plasma sterilization, gammaradiation, and peracetic acid sterilization. A sterilization techniquewhich does not significantly weaken the mechanical strength andbiotropic properties of the material is preferably used. For example, itis believed that strong gamma radiation can cause loss of strength inthe graft material. Example sterilization techniques include exposingthe graft to peracetic acid, low dose gamma irradiation, gas plasmasterilization, and high-pressure/supercritical carbon dioxide.

Further disclosed below are methods of sterilizing and decellularizingthe disclosed ECM compositions, whereby the methods not only do notsignificantly weaken the mechanical strength and bioptric properties ofthe ECM compositions, but also the methods are more effective indecellularizing the ECM compositions and in enhancing the incorporationof various additives into the ECM compositions. Thus, the disclosedsterilization and decellularization methods provide ECM compositionsthat are more decellularized and have a greater capacity to incorporateand then deliver more additives than ECM compositions known in the art.

ii. Synthetic ECM

Also disclosed are compositions comprising synthetic ECM for use in thedisclosed methods. Synthetic ECM for use in the disclosed compositionsand methods can be formed using synthetic molecules that polymerize muchlike native collagen and which form a scaffold environment that mimicsthe native environment of mammalian ECM scaffolds. Accordingly, suchmaterials as polyethylene terephthalate fiber (Dacron®),polytetrafluoroethylene (PTFE), glutaraldehyde-cross linked pericardium,polylactate (PLA), polyglycol (PGA), hyaluronic acid, polyethyleneglycol (PEG), polyethylene, nitinol, and collagen from non-animalsources (such as plants or synthetic collagens), can be used ascomponents of a synthetic extracellular matrix scaffold. The syntheticmaterials listed are standard in the art, and forming hydrogels andmatrix-like materials with them is also standard. Their effectivenesscan be tested in vivo as disclosed earlier, by testing in mammals, alongwith components that typically constitute native extracellular matrices,particularly the growth factors and cells responsive to them.

The extracellular matrix-like materials are described generally in Rossoet al. (Journal of Cellular Physiology 199:174-180, 2004), which isincorporated by reference herein for the teachings of how to make anduse these materials. In addition, some extracellular matrix-likematerials are listed here. Particularly useful biodegradable and/orbioabsorbable polymers include polylactides, polyglycolides,polycarprolactone, polydioxane and their random and block copolymers.Examples of specific polymers include poly D,L-lactide,polylactide-co-glycolide (85:15) and polylactide-co-glycolide (75:25).The biodegradable and/or bioabsorbable polymers used in the fibrousmatrix of the disclosed compositions and methods can have a molecularweight in the range of about 1,000 to about 8,000,000 g/mole, includingabout 4,000 to about 250,000 g/mole. The biodegradable and/orbioabsorbable fiberizable material can be a biodegradable andbioabsorbable polymer. Examples of suitable polymers can be found inBezwada, Rao S. et al. (1997) Poly(p-Dioxanone) and its copolymers, inHandbook of Biodegradable Polymers, A. J. Domb, J. Kost and D. M.Wiseman, editors, Hardwood Academic Publishers, The Netherlands, pp.29-61. The biodegradable and/or bioabsorbable polymer can contain amonomer selected from the group consisting of a glycolide, lactide,dioxanone, caprolactone, trimethylene carbonate, ethylene glycol andlysine. The material can be a random copolymer, block copolymer or blendof monomers, homopolymers, copolymers, and/or heteropolymers thatcontain these monomers. The biodegradable and/or bioabsorbable polymerscan contain bioabsorbable and biodegradable linear aliphatic polyesterssuch as polyglycolide (PGA) and its random copolymerpoly(glycolide-co-lactide-) (PGA-co-PLA). The FDA has approved thesepolymers for use in surgical applications, including medical sutures. Anadvantage of these synthetic absorbable materials is their degradabilityby simple hydrolysis of the ester backbone in aqueous environments, suchas body fluids. The degradation products are ultimately metabolized tocarbon dioxide and water or can be excreted via the kidneys. Thesepolymers are very different from cellulose-based materials, which cannotbe absorbed by the body.

Other examples of suitable biocompatible polymers are polyhydroxyalkylmethacrylates including ethylmethacrylate, and hydrogels such aspolyvinylpyrrolidone, polyacrylamides, etc. Other suitable bioabsorbablematerials are biopolymers which include collagen, gelatin, alginic acid,chitin, chitosan, fibrin, hyaluronic acid, dextran, polyamino acids,polylysine and copolymers of these materials. Any glycosaminoglycan(GAG) type polymer can be used. GAGs can include, e.g., heparin,chondroitin sulfate A or B, and hyaluronic acid, or their syntheticanalogues. Any combination, copolymer, polymer or blend thereof of theabove examples is contemplated for use according to the disclosedcompositions and methods. Such bioabsorbable materials can be preparedby known methods.

Nucleic acids from any source can be used as a polymeric biomaterial.Sources include naturally occurring nucleic acids as well as synthesizednucleic acids. Nucleic acids suitable for use in the disclosedcompositions and methods include naturally occurring forms of nucleicacids, such as DNA (including the A, B and Z structures), RNA (includingmRNA, tRNA, and rRNA together or separated), and cDNA, as well as anysynthetic or artificial forms of polynucleotides. The nucleic acids usedin the disclosed compositions and methods can be modified in a varietyof ways, including by cross linking, intra-chain modifications such asmethylation and capping, and by copolymerization. Additionally, otherbeneficial molecules can be attached to the nucleic acid chains. Thenucleic acids can have naturally occurring sequences or artificialsequences. The sequence of the nucleic acid can be irrelevant for manyaspects of the disclosure. However, special sequences can be used toprevent any significant effects due to the information coding propertiesof nucleic acids, to elicit particular cellular responses or to governthe physical structure of the molecule. Nucleic acids can be used in avariety of crystalline structures both in finished biomaterials andduring their production processes. Nucleic acid crystalline structurecan be influenced by salts used with the nucleic acid. For example, Na,K, Bi, and Ca salts of DNA all have different precipitation rates anddifferent crystalline structures. Additionally, pH influencescrystalline structure of nucleic acids.

The physical properties of the nucleic acids can also be influenced bythe presence of other physical characteristics. For example, inclusionof hairpin loops can result in more elastic biomaterials or can providespecific cleavage sites. The nucleic acid polymers and copolymersproduced can be used for a variety of tissue engineering applications,including to increase tissue tensile strength, improve wound healing,speed up wound healing, as templates for tissue formation, to guidetissue formation, to stimulate nerve growth, to improve vascularizationin tissues, as a biodegradable adhesive, as device or implant coating,or to improve the function of a tissue or body part. The polymers canalso more specifically be used as sutures, scaffolds and wounddressings. The type of nucleic acid polymer or copolymer used can affectthe resulting chemical and physical structure of the polymericbiomaterial.

iii. Combinations

The herein disclosed compositions can comprise combinations of mammalianECM from two or more sources or in two or more distinct forms. Thus, thedisclosed compositions can comprise any combination of native and/orsynthetic mammalian ECMs disclosed herein.

Thus, for example, the composition can comprise mammalian ECMcombinations from such sources as, for example, but not limited to,small intestine submucosa, liver basement membrane, stomach submucosa,urinary bladder submucosa, placental basement membrane, pancreaticbasement membrane, large intestine submucosa, lung interstitialmembrane, respiratory tract submucosa, heart extracellular matrix,dermal matrix, and in general extracellular matrix from any mammalianfetal tissue. Any one of these tissue sources can provide extracellularmatrix that can then be manipulated into a designated form (liquid,semi-solid, or solid form), for use in a composition.

The combinations of mammalian ECM from two or more sources can be mixedsolids, mixed liquids, mixed suspensions, mixed emulsions, mixed gels,mixed pastes, or mixed solid particulates. All of these compositions aremixtures of extracellular matrices from two or more sources, for examplemixtures of powders or particulates from two or more extracellularmatrices, mixtures of pastes from two or more extracellular matrices,mixtures of suspensions from two or more extracellular matrices,mixtures of emulsions or gels from two or more extracellular matricesand mixtures of liquids from two or more extracellular matrices.

The compositions can be made from three mammalian tissue sources, fourmammalian tissue sources, five mammalian tissue sources, six mammaliantissue sources, and conceivably up to ten or more tissue sources. Thesetissue sources can be from the same mammal (for example the same cow,the same pig, the same rodent, the same human, etc.), the same speciesof mammal (e.g. cow, pig, rodent, human), or different species ofmammals (for example liver matrix from a pig, small intestine submucosafrom a cow, and urinary bladder submucosa from a dog, all mixed togetherin the composition).

The compositions can comprise two or more liquid matrices (fromdifferent tissue sources) combined together. The composition can be twoor more emulsion matrices (from different tissue sources) combinedtogether. The composition can be two or more particulate matrices (fromdifferent tissue sources) combined together. The composition can be aliquid mixture of two or more extracellular matrices. The compositioncan be a suspension mixture of two or more extracellular matrices.

For example, a composition can comprise a combination of SIS in sheet,particulate, suspension, emulsion, gel or liquid form with SS, or LBM,or UBS in sheet, particulate, suspension, emulsion, gel or liquid form.For example, a composition can comprise a combination of SS in sheet,particulate, suspension, emulsion, gel or liquid form with SIS, or LBM,or UBS in sheet, particulate, suspension, emulsion, gel or liquid form.For example, a composition can comprise a combination of LBM in sheet,particulate, suspension, emulsion, gel or liquid form with SS, or SIS,or UBS in sheet, particulate, suspension, emulsion, gel or liquid form.For example, a composition can comprise a combination of UBS in sheet,particulate, suspension, emulsion, gel or liquid form with SS, or SIS,or LBM in sheet, particulate, suspension, emulsion, gel or liquid form.

The disclosed compositions can comprise combinations of mammalian ECMfrom one or more sources but in two or more distinct forms. For example,a composition can comprise a gel matrix combined with a particulatematrix. In some aspects, mammalian ECM in particulate form can be dustedonto mammalian ECM in a sheet form.

In some aspects, the composition can comprise a combination of SIS, SS,or LBM, or UBS in sheet, suspension, emulsion, gel or liquid form withSIS, SS, or LBM, or UBS in particulate four. In some aspects, thecomposition can comprise a combination of SIS, SS, or LBM, or UBS inparticulate, suspension, emulsion, gel or liquid form with SIS, SS, orLBM, or UBS in sheet form. In some aspects, the composition can comprisea combination of SIS, SS, or LBM, or UBS in sheet, particulate,suspension, gel or liquid form with SIS, SS, or LBM, or UBS in emulsionform. In some aspects, the composition can comprise a combination ofSIS, SS, or LBM, or UBS in sheet, particulate, suspension, emulsion, orliquid form with SIS, SS, or LBM, or UBS in gel form. In some aspects,the composition can comprise a combination of SIS, SS, or LBM, or UBS insheet, particulate, suspension, emulsion, or gel form with SIS, SS, orLBM, or UBS in liquid form. In some aspects, the composition cancomprise a combination of SIS, SS, or LBM, or UBS in sheet, particulate,liquid, emulsion, or gel form with SIS, SS, or LBM, or UBS in suspensionform.

As disclosed herein, the composition comprising mammalian ECM can beprepared for preferred consistency. For example, mammalian ECM can beprepared as a combination of gel and particulate in a ratio optimal toprevent dissipation into the blood stream. For example, the compositioncomprising mammalian ECM can comprise about 40% ECM in gel form andabout 60% ECM in dry particulate form. Thus, disclosed herein is acomposition comprising mammalian ECM in both gel and dry particulateforms, wherein the gel form comprises about 10, 15, 20, 25, 30, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 50% of the ECM in the composition.Thus, the dry particulate form can comprise about 90, 85, 80, 75, 70,65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 50% of the ECM in thecomposition.

Selection of the concentrations of the liquid or semi-solid compositions(liquids, gels, suspensions emulsions, or pastes) is important. Forexample, the liquid forms can be present in a range of concentrations,from very dilute at about 0.001 mg/ml to greater concentrations of up toabout 200 mg/ml. The concentrations can further be found in morespecific ranges such as, for example, the following set of ranges: fromabout 5 mg/ml to about 150 mg/ml, from about 10 mg/ml to about 125mg/ml, from about 25 mg/ml to about 100 mg/ml, from about 20 mg/ml toabout 75 mg/ml, from about 25 mg/ml to about 60 mg/ml, from about 30mg/ml to about 50 mg/ml, from about 35 mg/ml to about 45 mg/ml, and fromabout 40 mg/ml to about 42 mg/ml. This set of ranges is exemplary andnot intended to be exhaustive. It is contemplated that any value withinany of these specifically listed ranges is a reasonable and useful valuefor a concentration of a liquid or semi-solid component of thecomposition.

The emulsion can be more concentrated than a liquid form and can retaina shape which can be useful in applying the matrix composition tocertain parts of the body, hence its characterization as a “semi-solid”.The emulsion can be concentrated enough to form shapes like a plug orother configuration suited to the site at which the matrix compositionis being applied. Thick emulsion can be painted or otherwise applied ata site as a paste, and dusted with solid particulate on top of theemulsion. The solid particulate can be reconstituted to form theemulsion, or can be applied dry as a particulate powder which can dust aregion in the subject being treated

Dry particulate or reconstituted particulate that forms an emulsion oftwo or more mammalian ECM can be mixed together in some proportion. Forexample, 50% of SIS can be mixed with 50% of SS in a vial. This mixturecan then be fluidized by hydrating it in a suitable buffer, for examplesaline. The hydration can be accomplished to a desired concentration ofthe mammalian ECM mixture, for example in a range from about 0.001 mg/mlto about 200 mg/ml. The concentrations can further be found in morespecific ranges such as for example the following set of ranges: fromabout 5 mg/ml to about 150 mg/ml, from about 10 mg/ml to about 125mg/ml, from about 25 mg/ml to about 100 mg/ml, from about 20 mg/ml toabout 75 mg/ml, from about 25 mg/ml to about 60 mg/ml, from about 30mg/ml to about 50 mg/ml, from about 35 mg/ml to about 45 mg/ml, and fromabout 40 mg/ml. to about 42 mg/ml. This set of ranges is exemplary andnot intended to be exhaustive. It is contemplated that any value withinany of these specifically listed ranges is a reasonable and useful valuefor a concentration of a liquid or semi-solid component of thecomposition.

The lower the concentration of extracellular matrix the more liquid thecomposition will be. The higher the concentration of extracellularmatrix the more the composition approaches a gel-like emulsion orsemi-solid consistency.

The ratio of the mixtures of the two (or more) extracellular matrices inany given composition from different sources (or the same source) can beunequal. So for example, LBM can be present at 75% and SIS can bepresent at 25%, i.e., a 3:1 ratio). Any suitable ratio can be used: 1:1,1:2, 1:3, 1:4, 1:5, and so on. Where 3 or more tissue sources ofextracellular matrix are represented in the composition, the same typeof balance or imbalance in the amounts of the matrices can occur. Forexample, for extracellular matrix from 3 sources, each source can bepresent in equal proportions, i.e., 1:1:1 (33%/33%/33%). Alternatively,a disproportionate amount of the particulate can be from one source,e.g., 2:1:1 (50%/25%/25%). Likewise, all three sources can be present indisproportionate amounts, e.g., 50%/30%/20%.

The two or more mammalian ECMs can be fluidized (or emulsified)separately and the fluidized or emulsified compositions mixed together.As another alternative, the two or more mammalian ECMs can be fluidizedor emulsified separately and administered separately. In addition, thetwo or more mammalian ECMs can remain in particulate solid form and bemixed together in a vial for administration as a solid combinationparticulate. Rehydration of a dry particulate mammalian ECM mixture canbe accomplished just prior to use.

2. Proteins

The disclosed compositions comprising mammalian ECM can further compriseexogenous proteins, such as those normally found in mammalian ECM. Theprotein can be a collagen, a proteoglycan, a glycosaminoglycan (GAG)chain, a glycoprotein, a growth factor, a cytokine, a cell-surfaceassociated protein, a cell adhesion molecule (CAM), an angiogenic growthfactor, an endothelial ligand, a matrikine, a matrix metalloprotease, acadherin, an immunoglobulin, a fibril collagen, a non-fibrillarcollagen, a basement membrane collagen, a multiplexin, a small-leucinerich proteoglycan, decorin, biglycan, a fibromodulin, keratocan,lumican, epiphycan, a heparan sulfate proteoglycan, perlecan, agrin,testican, syndecan, glypican, serglycin, selectin, a lectican, aggrecan,versican, neurocan, brevican, cytoplasmic domain-44 (CD-44), macrophagestimulating factor, amyloid precursor protein, heparin, chondroitinsulfate B (dermatan sulfate), chondroitin sulfate A, heparan sulfate,hyaluronic acid, fibronectin (Fn), tenascin, elastin, fibrillin,laminin, nidogen/entactin, fibulin I, fibulin II, integrin, atransmembrane molecule, platelet derived growth factor (PDGF), epidermalgrowth factor (EGF), transforming growth factor alpha (TGF-alpha),transforming growth factor beta (TGF-β), fibroblast growth factor-2(FGF-2) (also called basic fibroblast growth factor (bFGF)),thrombospondin, osteopontin, angiotensin converting enzyme (ACE), or avascular endothelial growth factor (VEGF). This list is not intended tobe exhaustive.

Thus, the herein disclosed compositions comprising a mammalian ECM cancomprise collagen I and III, elastin, laminin, CD44, hyaluronan,syndecan, bFGF, HGF, PDGF, VEGF, Fn, tenascin, heparin, heparan sulfate,chondroitin sulfate B, integrins, decorin, TGF-β, or a combinationthereof.

2. Cells

In some aspects, the herein disclosed compositions comprising mammalianECM further comprise one or more cells. In some aspects the cells arenon-native, i.e., heterologous to the mammalian ECM. In some aspects thecells are autologous. In some aspects, the cells are stem cells. Anon-exhaustive list of stem cells includes a human embryonic stem cell,a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, anautotransplanted expanded cardiomyocyte, an adipocyte, a totipotentcell, a pluripotent cell, a blood stem cell, a myoblast, an adult stemcell, a bone marrow cell, a mesenchymal cell, an embryonic stem cell, aparenchymal cell, an epithelial cell, an endothelial cell, a mesothelialcell, a fibroblast, an osteoblast, a chondrocyte, an exogenous cell, anendogenous cell, a stem cell, a hematopoietic stem cell, a pluripotentstem cell, a bone marrow-derived progenitor cell, a progenitor cell, amyocardial cell, a skeletal cell, a fetal cell, an embryonic cell, anundifferentiated cell, a multi-potent progenitor cell, a unipotentprogenitor cell, a monocyte, a cardiomyocyte, a cardiac myoblast, askeletal myoblast, a macrophage, a capillary endothelial cell, axenogenic cell, an allogenic cell, an adult stem cell, and a post-natalstem cell.

In some aspects, the stem cells have the potential to differentiate intocardiac tissue cells. Thus, in some aspects, the stem cells arepluripotent. In some aspects, the stem cells are angioblasts orhemangioblasts. In some aspects, the stem cells are myoblasts. Stemcells can be derived and maintained using standard methods for stem cellculture.

2. Pharmaceuticals

The herein disclosed compositions comprising mammalian ECM can furthercomprise any known or newly discovered substance that can beadministered to the heart of a subject. For example, the hereindisclosed compositions comprising mammalian ECM can further comprise anantiarrhythmic agent. Antiarrhythmic agents are a group ofpharmaceuticals that are used to suppress fast and/or irregular rhythmsof the heart (cardiac arrhythmias).

The Vaughan Williams classification, introduced in 1970, is one of themost widely used classification schemes for antiarrhythmic agents. Thisscheme classifies a drug based on the primary mechanism of itsantiarrhythmic effect. There are five main classes in the VaughanWilliams classification of antiarrhythmic agents: Class I agentsinterfere with the sodium (Na⁺) channel; Class II agents areanti-sympathetic nervous system agents (most agents in this class arebeta blockers); Class III agents affect potassium (K⁺) efflux; Class IVagents affect calcium channels and the AV node; and Class V agents workby other or unknown mechanisms.

Class Ia agents include Quinidine, Procainamide, and Disopyramide. ClassIb agents include Lidocaine, Phenyloin, and Mexiletine. Class Ic agentsinclude Flecamide, Propafenone, and Moricizine. Class II agents includePropranolol, Esmolol, Timolol, Metoprolol, and Atenolol. Class IIIagents include Amiodarone, Sotalol, Ibutilide, and Dofetilide. Class IVagents include Verapamil, and Diltiazem. Class V agents includeAdenosine and Digoxin.

Thus, the herein disclosed compositions comprising mammalian ECM canfurther comprise one or more of Quinidine, Procainamide, Disopyramide,Lidocaine, Phenyloin, Mexiletine, Flecamide, Propafenone, Moricizine,Propranolol, Esmolol, Timolol, Metoprolol, Atenolol, Amiodarone,Sotalol, Ibutilide, Dofetilide, Verapamil, Diltiazem, Adenosine andDigoxin.

The provided compositions can further comprise one or more antibiotics(e.g., Aminoglycosides, Cephalosporins, Chloramphenicol, Clindamycin,Erythromycins, Fluoroquinolones, Macrolides, Azolides, Metronidazole,Penicillins, Tetracyclines, Trimethoprim-sulfamethoxazole, andVancomycin).

The provided compositions can further comprise one or more steroids(e.g., Andranes (e.g., Testosterone), Cholestanes (e.g., Cholesterol),Cholic acids (e.g., Cholic acid), Corticosteroids (e.g., Dexamethasone),Estraenes (e.g., Estradiol), and Pregnanes (e.g., Progesterone).

The provided compositions can further comprise one or more classes ofnarcotic and non-narcotic analgesics, including, but not limited to,Morphine, Codeine, Heroin, Hydromorphone, Levorphanol, Meperidine,Methadone, Oxydone, Propoxyphene, Fentanyl, Methadone, Naloxone,Buprenorphine, Butorphanol, Nalbuphine, and Pentazocine.

The provided compositions can further comprise one or moreanti-inflammatory agents, including, but not limited to, Alclofenac,Alclometasone Dipropionate, Algestone Acetonide, alpha Amylase,Amcinafal, Amcinafide, Amfenac Sodium, Amiprilose Hydrochloride,Anakinra, Anirolac, Anitrazafen, Apazone, Balsalazide Disodium,Bendazac, Benoxaprofen, Benzydamine Hydrochloride, Bromelains,Broperamole, Budesonide, Carprofen, Cicloprofen, Cintazone, Cliprofen,Clobetasol Propionate, Clobetasone Butyrate, Clopirac, CloticasonePropionate, Cormethasone Acetate, Cortodoxone, Decanoate, Deflazacort,Delatestryl, Depo-Testosterone, Desonide, Desoximetasone, DexamethasoneDipropionate, Diclofenac Potassium, Diclofenac Sodium, DiflorasoneDiacetate, Diflumidone Sodium, Diflunisal, Difluprednate, Diftalone,Dimethyl Sulfoxide, Drocinonide, Endrysone, Enlimomab, Enolicam Sodium,Epirizole, Etodolac, Etofenamate, Felbinac, Fenamole, Fenbufen,Fenclofenac, Fenclorac, Fendosal, Fenpipalone, Fentiazac, Flazalone,Fluazacort, Flufenamic Acid, Flumizole, Flunisolide Acetate, Flunixin,Flunixin Meglumine, Fluocortin Butyl, Fluorometholone Acetate,Fluquazone, Flurbiprofen, Fluretofen, Fluticasone Propionate,Furaprofen, Furobufen, Halcinonide, Halobetasol Propionate, HalopredoneAcetate, Ibufenac, Ibuprofen, Ibuprofen Aluminum, Ibuprofen Piconol,Ilonidap, Indomethacin, Indomethacin Sodium, Indoprofen, Indoxole,Intrazole, Isoflupredone Acetate, Isoxepac, Isoxicam, Ketoprofen,Lofemizole Hydrochloride, Lomoxicam, Loteprednol Etabonate,Meclofenamate Sodium, Meclofenamic Acid, Meclorisone Dibutyrate,Mefenamic Acid, Mesalamine, Meseclazone, Mesterolone,Methandrostenolone, Methenolone, Methenolone Acetate, MethylprednisoloneSuleptanate, Morniflumate, Nabumetone, Nandrolone, Naproxen, NaproxenSodium, Naproxol, Nimazone, Olsalazine Sodium, Orgotein, Orpanoxin,Oxandrolane, Oxaprozin, Oxyphenbutazone, Oxymetholone, ParanylineHydrochloride, Pentosan Polysulfate Sodium, Phenbutazone SodiumGlycerate, Pirfenidone, Piroxicam, Piroxicam Cinnamate, PiroxicamOlamine, Pirprofen, Prednazate, Prifelone, Prodolic Acid, Proquazone,Proxazole, Proxazole Citrate, Rimexolone, Romazarit, Salcolex,Salnacedin, Salsalate, Sanguinarium Chloride, Seclazone, Sermetacin,Stanozolol, Sudoxicam, Sulindac, Suprofen, Talmetacin, Talniflumate,Talosalate, Tebufelone, Tenidap, Tenidap Sodium, Tenoxicam, Tesicam,Tesimide, Testosterone, Testosterone Blends, Tetrydamine, Tiopinac,Tixocortol Pivalate, Tolmetin, Tolmetin Sodium, Triclonide,Triflumidate, Zidometacin, and Zomepirac Sodium.

The provided compositions can further comprise one or morelipid-lowering drugs. As used herein, the term “lipid-lowering drug”refers to a drug that can be administered to a subject to reduce theserum levels of various heart disease-associated lipids, including, butnot limited to, cholesterol, low-density lipoprotein (LDL), verylow-density lipoprotein (VLDL), and triglycerides.

For example, the lipid-lowering drugs can be statins including, but notlimited to, Lovastatin, Simvastatin, Atorvastatin, Fluvastatin,Pravastatin, Rosuvastatin, Cervistatin, and Pitavastatin. It iscontemplated that any statin drug, now known or developed in the future,having lipid-reducing and/or anti-inflammatory properties can be used inthe compositions described herein.

The provided compositions can further comprise one or moreanti-histaminic agents, including, but not limited to, Ethanolamines(like diphenhydramine carbinoxamine), Ethylenediamine (liketripelennamine pyrilamine), Alkylamine (like chlorpheniramine,dexchlorpheniramine, brompheniramine, triprolidine), astemizole,loratadine, fexofenadine, Bropheniramine, Clemastine, Acetaminophen,Pseudoephedrine, and Triprolidine.

The provided compositions can further comprise one or moreantineoplastic drugs, including, but not limited to, Acivicin,Aclarubicin, Acodazole Hydrochloride, AcrQnine, Adozelesin, Aldesleukin,Altretamine, Ambomycin, Ametantrone Acetate, Aminoglutethimide,Amsacrine, Anastrozole, Anthramycin, Asparaginase, Asperlin,Azacitidine, Azetepa, Azotomycin, Batimastat, Benzodepa, Bicalutamide,Bisantrene Hydrochloride, Bisnafide Dimesylate, Bizelesin, BleomycinSulfate, Brequinar Sodium, Bropirimine, Busulfan, Cactinomycin,Calusterone, Caracemide, Carbetimer, Carboplatin, Carmustine, CarubicinHydrochloride, Carzelesin, Cedefingol, Chlorambucil, Cirolemycin,Cisplatin, Cladribine, Crisnatol Mesylate, Cyclophosphamide, Cytarabine,Dacarbazine, Dactinomycin, Daunorubicin Hydrochloride, Decitabine,Dexormaplatin, Dezaguanine, Dezaguanine Mesylate, Diaziquone, Docetaxel,Doxorubicin, Doxorubicin Hydrochloride, Droloxifene, DroloxifeneCitrate, Dromostanolone Propionate, Duazomycin, Edatrexate, EflomithineHydrochloride, Elsamitrucin, Enloplatin, Enpromate, Epipropidine,Epirubicin Hydrochloride, Erbulozole, Esorubicin Hydrochloride,Estramustine, Estramustine Phosphate Sodium, Etanidazole, Ethiodized OilI 131, Etoposide, Etoposide Phosphate, Etoprine, FadrozoleHydrochloride, Fazarabine, Fenretinide, Floxuridine, FludarabinePhosphate, Fluorouracil, Fluorocitabine, Fosquidone, Fostriecin Sodium,Gemcitabine, Gemcitabine Hydrochloride, Gold Au 198, Hydroxyurea,Idarubicin Hydrochloride, Ifosfamide, Ilmofosine, Interferon Alfa-2a,Interferon Alfa-2b, Interferon Alfa-n1, Interferon Alfa-n3, InterferonBeta-I a, Interferon Gamma-Ib, Iproplatin, Irinotecan Hydrochloride,Lanreotide Acetate, Letrozole, Leuprolide Acetate, LiarozoleHydrochloride, Lometrexol Sodium, Lomustine, Losoxantrone Hydrochloride,Masoprocol, Maytansine, Mechlorethamine Hydrochloride, MegestrolAcetate, Melengestrol Acetate, Melphalan, Menogaril, Mercaptopurine,Methotrexate, Methotrexate Sodium, Metoprine, Meturedepa, Mitindomide,Mitocarcin, Mitocromin, Mitogillin, Mitomalcin, Mitomycin, Mitosper,Mitotane, Mitoxantrone Hydrochloride, Mycophenolic Acid, Nocodazole,Nogalamycin, Ormaplatin, Oxisuran, Paclitaxel, Pegaspargase, Peliomycin,Pentamustine, Peplomycin Sulfate, Perfosfamide, Pipobroman, Piposulfan,Piroxantrone Hydrochloride, Plicamycin, Plomestane, Porfimer Sodium,Porfiromycin, Prednimustine, Procarbazine Hydrochloride, Puromycin,Puromycin Hydrochloride, Pyrazofurin, Riboprine, Rogletimide, Safmgol,Safingol Hydrochloride, Semustine, Simtrazene, Sparfosate Sodium,Sparsomycin, Spirogermanium Hydrochloride, Spiromustine, Spiroplatin,Streptonigrin, Streptozocin, Strontium Chloride Sr 89, Sulofenur,Talisomycin, Taxane, Taxoid, Tecogalan Sodium, Tegafur, TeloxantroneHydrochloride, Temoporfin, Teniposide, Teroxirone, Testolactone,Thiamiprine, Thioguanine, Thiotepa, Tiazofurin, Tirapazamine, TopotecanHydrochloride, Toremifene Citrate, Trestolone Acetate, TriciribinePhosphate, Trimetrexate, Trimetrexate Glucuronate, Triptorelin,Tubulozole Hydrochloride, Uracil Mustard, Uredepa, Vapreotide,Verteporfin, Vinblastine Sulfate, Vincristine Sulfate, Vindesine,Vindesine Sulfate, Vinepidine Sulfate, Vinglycinate Sulfate,Vinleurosine Sulfate, Vinorelbine Tartrate, Vinrosidine Sulfate,Vinzolidine Sulfate, Vorozole, Zeniplatin, Zinostatin, and ZorubicinHydrochloride.

The herein provided compositions can further comprise one or moreradiosensitizers including, but not limited to, gemcitabine,5-fluorouracil, pentoxifylline, and vinorelbine.

2. Carriers

The disclosed mammalian ECM can be combined, conjugated or coupled withor to carriers and other compositions to aid administration, delivery orother aspects of the ECM and their use. For convenience, suchcompositions are referred to herein as carriers. Carriers can, forexample, be a small molecule, pharmaceutical drug, fatty acid,detectable marker, conjugating tag, nanoparticle, or enzyme.

The disclosed compositions can be used therapeutically in combinationwith a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material can be administered to a subject, alongwith the composition, without causing any undesirable biological effectsor interacting in a deleterious manner with any of the other componentsof the pharmaceutical composition in which it is contained. The carrierwould naturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of the pharmaceuticallyacceptable carrier include, but are not limited to, saline, Ringer'ssolution and dextrose solution. The pH of the solution is preferablyfrom about 5 to about 8, and more preferably from about 7 to about 7.5.Further carriers include sustained release preparations such assemipermeable matrices of solid hydrophobic polymers containing thecomposition, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for example, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH.

Pharmaceutical compositions can include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions can also includeone or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders can be desirable.

Some of the compositions can potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

D. METHODS OF MAKING THE COMPOSITIONS

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted. For example, U.S. Pat. No. 5,275,826, U.S.Pat. No. 5,554,389, U.S. Pat. No. 6,099,567, and U.S. Pat. No.6,379,710, are disclosed herein by reference for methods of makingcompositions comprising small intestine submucosa (SIS), urinary bladdersubmucosa (UBS), stomach submucosa (SS), and liver basement membrane(LBM), respectively.

E. METHODS OF STERILIZING THE COMPOSITIONS

Unless otherwise expressly stated, it is in no way intended that anymethod or aspect set forth herein be construed as requiring that itssteps be performed in a specific order. Accordingly, where a methodclaim does not specifically state in the claims or descriptions that thesteps are to be limited to a specific order, it is in no way intendedthat an order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including matters of logic withrespect to arrangement of steps or operational flow, plain meaningderived from grammatical organization or punctuation, or the number ortype of aspects described in the specification.

As used herein, the term “acellular” is meant to describe extracellularmatrix compositions that are at least 80% decellularized such that theextracellular matrix composition is 80% without cells and/or cellularremnants. In some exemplary aspects described herein, the term“acellular” can refer to extracellular matrix compositions that are atleast 90% decellularized such that the extracellular matrix compositionis at least 90% without cells and/or cellular remnants. In otherexemplary aspects described herein, the term “acellular” can refer toextracellular matrix compositions that are at least 95% decellularizedsuch that the extracellular matrix composition is at least 95% withoutcells and/or cellular remnants. In other exemplary aspects describedherein, the term “acellular” can refer to extracellular matrixcompositions that are at least 96% decellularized such that theextracellular matrix composition is at least 96% without cells and/orcellular remnants. In still other exemplary aspects described herein,the term “acellular” can refer to extracellular matrix compositions thatare at least 97% decellularized such that the extracellular matrixcomposition is at least 97% without cells and/or cellular remnants. Infurther exemplary aspects described herein, the term “acellular” canrefer to extracellular matrix compositions that are at least 98%decellularized such that the extracellular matrix composition is atleast 98% without cells and/or cellular remnants. In still furtherexemplary aspects described herein, the term “acellular” can refer toextracellular matrix compositions that are at least 99% decellularizedsuch that the extracellular matrix composition is at least 99% withoutcells and/or cellular remnants. Thus, as used herein, the term“acellular” can refer to extracellular matrix compositions that aredecellularized at levels of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, and anypercentages falling between these values.

As used herein, the term “additive” refers to materials that can beselectively incorporated into the disclosed ECM materials to impartpredetermined properties to the sterilized, acellular ECM compositionsdisclosed herein. Such additives can include, for example and withoutlimitation, growth factors, cytokines, proteoglycans, glycosaminoglycans(GAGS), proteins, peptides, nucleic acids, small molecules, cells andpharmaceutical agents, such as statin drugs, corticosterioids,anti-arrhythmic drugs, nonsteroidal anti-inflammatory drugs, otheranti-inflammatory compounds, nanoparticles, and metallic compounds.

As used herein, the term “contemporaneously” refers to the simultaneousand/or overlapping occurrence of events, as well as the sequentialoccurrence of events within about thirty minutes before or after oneanother. Thus, if a first event occurs, then a second event can be saidto have occurred contemporaneously with the first event if it occurredconcurrently with the first event or within thirty minutes before orafter the first event. For example, if a first method step is performed,then a second method step performed five minutes after the first methodstep can be said to be performed “contemporaneously” with the firstmethod step. Similarly, if the second method step was performed tenminutes before performance of a third method step, then the secondmethod step can be said to be performed “contemporaneously” with thethird method step.

As used herein, the term “emulsion” refers to a mixture in which a firstECM material is dispersed within a second ECM material, with the firstECM material being immiscible with the second ECM material. The“emulsions” described herein can refer to either oil-in-water typeemulsions or water-in-oil type emulsions.

As used herein, the term “suspension” refers to a mixture in which asolid ECM material, such as, for example and without limitation,particulate ECM, is dispersed (suspended) in a fluid ECM material, suchas, for example and without limitation, ECM gel or ECM liquid.

As used herein, the term “supercritical” refers to a fluid state of amaterial when it is held at or above its critical temperature andcritical pressure. When a material is held at or above its criticaltemperature and critical pressure, then it typically adopts functionalproperties of both a gas and a liquid and is said to function as asupercritical fluid. Thus, for example, when carbon dioxide is held ator above its critical temperature (31.1° C.) and its critical pressure(1,071 psi), it behaves as a supercritical carbon dioxide fluid and can,for example, exhibit the expansion properties of a gas while having thedensity of a liquid.

Described herein are sterilized, acellular extracellular matrix (ECM)compositions and methods for making such compositions. As describedherein, the disclosed extracellular matrix compositions are formed bycontemporaneously sterilizing and decellularizing an isolated ECMmaterial. More particularly, the disclosed methods contemporaneouslyaccomplish desired sterilization and decellularization of the isolatedECM material such that the native properties of the tissue compositionare maintained and the ECM material is rendered sterile and acellular.

As further described herein, the disclosed methods make use of rapiddepressurization of the ECM material to decellularize the ECM material.This rapid depressurization of the ECM material occurs atdepressurization rates that are significantly higher than thedepressurization rates applied in previously known methods. In additionto decellularizing the ECM material as described herein, the rapiddepressurization of the ECM material also can be used to enhance theincorporation of desired sterilants and additives into the ECM material.

ECM Compositions

In exemplary aspects, a sterilized, acellular ECM composition cancomprise any known ECM component or material, including, for example andwithout limitation, mucosal layers and components, submucosal layers andcomponents, muscularis layers and components, and/or basement membranelayers and components. It is contemplated that a disclosed sterilized,acellular ECM composition can comprise an ECM material obtained from anymammalian tissue source, including, for example and without limitation,stomach tissue (e.g., stomach submucosa (SS)), small intestinal tissue(e.g., small intestinal submucosa (SIS)), large intestinal tissue,bladder tissue (e.g., urinary bladder submucosa (UBS)), liver tissue(e.g., liver basement membrane (LBM)), heart tissue (e.g., pericardium),lung tissue, kidney tissue, pancreatic tissue, prostate tissue,mesothelial tissue, fetal tissue, a placenta, a ureter, veins, arteries,tissue surrounding the roots of developing teeth, and tissue surroundinggrowing bone. It is further contemplated that a disclosed sterilized,acellular ECM composition can comprise an ECM material obtained from ECMcomponents or materials of one or more mammals including, for exampleand without limitation, humans, cows, pigs, dogs, sheep, cats, horses,rodents, and the like. Thus, it is contemplated that a disclosedsterilized, acellular ECM composition can comprise ECM components ormaterials from two or more of the same mammalian species, such as, forexample and without limitation, two or more cows, two or more pigs, twoor more dogs, or two or more sheep. It is further contemplated that adisclosed sterilized, acellular ECM composition can comprise ECMcomponents or materials from two or more different mammalian species,such as, for example and without limitation, a pig and a cow, a pig anda dog, a pig and a sheep, or a cow and a sheep. It is still furthercontemplated that a disclosed sterilized, acellular ECM composition cancomprise ECM components or materials obtained from a first tissuesource, such as, for example and without limitation, SIS, from a firstmammal, as well as ECM components or materials obtained from a secondtissue source, such as, for example and without limitation, SS, from asecond mammal.

In various aspects, a disclosed sterilized, acellular ECM compositioncan be produced in any suitable shape, including, for example andwithout limitation, a substantially flat sheet, a cylindrical tube, asubstantially spherical structure, or a multi-laminate structure. It iscontemplated that a disclosed sterilized, acellular ECM composition canalso be produced in any suitable form, including, for example andwithout limitation, a solid, liquid, gel, particulate, sponge, emulsion,or suspension form. In one exemplary aspect, it is contemplated that adisclosed sterilized, acellular ECM composition can comprise an outerlayer of solid ECM material that encloses an inner layer of liquid,particulate, emulsion, suspension, and/or gel ECM material.

In another exemplary aspect, it is contemplated that a disclosedsterilized, acellular ECM composition can comprise one or more types ofparticulate ECM materials that are suspended within an ECM gel to forman ECM suspension. In this aspect, it is contemplated that theparticulates within a disclosed ECM suspension can have a diameterranging from about 5 μm to about 300 μm, with an average diameterranging from about 90 μm to about 100 μm. It is further contemplatedthat the percentage of gel within a disclosed ECM suspension can rangefrom about 5% to about 50%, while the percentage of particulate within adisclosed ECM suspension can range from about 50% to about 95%. Thus, itis contemplated that the percentage of gel within a disclosed ECMsuspension can be about 10%, while the percentage of particulate withinthe ECM suspension can be about 90%. It is further contemplated that thepercentage of gel within a disclosed ECM suspension can be about 15%,while the percentage of particulate within the ECM suspension can beabout 85%. More preferably, the percentage of gel within a disclosed ECMsuspension can range from about 20% to about 30%, while the percentageof particulate within a disclosed ECM suspension can range from about70% to about 80%. Thus, in an exemplary aspect, the percentage of gelwithin a disclosed ECM suspension can be about 20%, while the percentageof particulate within the ECM suspension can be about 80%. In anotherexemplary aspect, the percentage of gel within a disclosed ECMsuspension can be about 25%, while the percentage of particulate withinthe ECM suspension can be about 75%. In an additional exemplary aspect,the percentage of gel within a disclosed ECM suspension can be about30%, while the percentage of particulate within the ECM suspension canbe about 70%. Although the above ranges refer to particular beginningpoint values and end point values, it is contemplated that a disclosedECM suspension can be formed from gel percentages and particulatepercentages falling within any of the ranges disclosed above.

In a further aspect, it is contemplated that a disclosed ECM suspensioncan comprise sterilized, decellularized ECM. In exemplary aspects, theECM gel of a disclosed ECM suspension can be a hydrolyzed ECM. In theseaspects, it is contemplated that the ECM gel of a disclosed ECMsuspension can comprise ECM that is greater than about 50% hydrolyzed,more preferably, greater than about 70% hydrolyzed, and, mostpreferably, greater than about 90% hydrolyzed. In one exemplary aspect,the ECM gel of a disclosed ECM suspension can comprise ECM that is about100% hydrolyzed. It is still further contemplated that the ECMcomponents of the suspension can comprise at least one of:glycoproteins, such as, for example and without limitation, fibronectinand laminin; glycosaminoglycans, such as, for example and withoutlimitation, heparan, hyaluronic acid, and chondroitin sulfate; andgrowth factors, thereby providing additional bioavailability for nativecellular components. It is contemplated that the ECM components of thesuspension can provide a structural and biochemical microenvironmentthat promotes cell growth and stem cell attraction followingimplantation of a disclosed ECM suspension within a subject. It isfurther contemplated that the ECM gel of a disclosed ECM suspension canfunction as a bulking agent that preserves a desired biomechanicalenvironment until the cells of the subject can begin producing their ownECM.

It is still further contemplated that the desired biomechanicalenvironment that is preserved by the ECM gel can substantiallycorrespond to a biomechanical environment in native tissue. Thus, it iscontemplated that the ECM gel of a disclosed ECM suspension can have anelastic modulus that is substantially equal to the elastic modulus of atarget site within a subject. In exemplary aspects, the elastic modulusof the ECM gel of a disclosed ECM suspension can range from about 5 kPato about 50 kPa, and, more preferably, from about 10 kPa to about 15kPa.

In one non-limiting exemplary aspect, it is contemplated that, when adisclosed ECM suspension is configured for injection at a target site onor within the heart of a subject, the elastic modulus of the ECM gel ofthe disclosed ECM suspension can be about 11.5 kPa, which is the elasticmodulus of cardiac muscle. As used herein, the term “on or within theheart” refers to locations that are, for example and without limitation,on or within the pericardium, epicardium, myocardium, endocardium,ventricles, atria, aorta, pulmonary arteries, pulmonary veins, venacavae, and the like. In another aspect, it is further contemplated thata disclosed ECM suspension can be injected at a target site on or withinthe heart of the subject to therapeutically prevent or reverse leftventricular wall negative remodeling that occurs following acutemyocardial infarction and/or chronic coronary heart disease. As usedherein, the term “negative remodeling” refers to the detrimental and/orundesired changes in the heart that occur in response to myocardialinjury; such undesired changes include, for example and withoutlimitation, alterations in myocyte biology, myocyte loss, extracellularmatrix degradation, extracellular matrix replacement fibrosis,alterations in left ventricular chamber geometry, increased wall stress(afterload), afterload mismatch, episodic subendocardial hypoperfusion,increased oxygen utilization, sustained hemodynamic overloading, andworsening activation of compensatory mechanisms. It is still furthercontemplated that a disclosed ECM suspension can be injected at a targetsite on or within the heart of the subject to therapeutically treatheart failure.

In an exemplary aspect, it is contemplated that a disclosed ECMsuspension can be injected at a target site on or within the heart of asubject, such as, for example and without limitation, on or within thepericardium, epicardium, myocardium, endocardium, ventricles, atria,aorta, and the like. Optionally, in one aspect, a disclosed ECMsuspension can be injected in a grid-like pattern. In this aspect, it iscontemplated that a disclosed ECM suspension can be injected as a firstseries of spaced, substantially parallel lines and a second series ofspaced, substantially parallel lines that are substantiallyperpendicular to the first series of spaced, substantially parallellines, thereby defining the grid-like pattern.

In another aspect, it is contemplated that a disclosed ECM suspensioncan be applied to a target site on or within the heart of a subject tocreate a film of a disclosed ECM suspension having a thickness rangingfrom about 0.1 mm to about 10 mm, more preferably, from about 1 mm toabout 5 mm, and, most preferably, from about 2 mm to about 4 mm. In oneexemplary aspect, it is contemplated that a disclosed ECM suspension canbe applied to a target site on or within the heart of the subject tocreate a film of the ECM suspension having a thickness of about 3 mm.

In a further exemplary aspect, it is contemplated that a disclosed ECMsuspension can be injected at a target site positioned within themyocardium or scar tissue of the heart of a subject. In this aspect, itis contemplated that a disclosed ECM suspension can be injected into themyocardium or scar tissue within the heart of the subject at a desireddepth relative to an outer surface of the pericardium. It is furthercontemplated that the desired depth at which a disclosed ECM suspensionis injected can range from about 0.5 mm to about 5 mm, more preferably,from about 1 mm to about 3 mm, and most preferably, from about 1.5 mm toabout 2.5 mm. In one exemplary aspect, it is contemplated that thedesired depth at which a disclosed ECM suspension is injected can beabout 2 mm. In this aspect, it is contemplated that the desired depth atwhich a disclosed ECM suspension is injected can correspond to aposition proximate the junction between the epicardium and themyocardium. It is further contemplated that the desired depth at which adisclosed ECM suspension is injected can correspond to a positionproximate ischemic and/or inflamed and/or injured heart tissue. In anexemplary aspect, it is contemplated that the desired depth at which adisclosed ECM suspension is injected can correspond to a positionproximate necrotic and/or infarcted myocardium.

In exemplary aspects, when a disclosed ECM suspension is to be injectedat a target site within the myocardium and/or one or more chambers ofthe heart of a subject following the occurrence of a myocardialinfarction, it is contemplated that the ECM suspension should beinjected at the target site during one of two possible time periods:prior to full onset of the inflammatory response of the subject or afterthe inflammatory response of the subject has decreased. In one aspect,when the ECM suspension is injected at the target site prior to fullonset of the inflammatory response of the subject, it is contemplatedthat the ECM suspension should be injected at the target sitesubstantially immediately after occurrence of the myocardial infarctionup to the time of therapeutic reperfusion and/or revascularization ofthe heart (using, for example, a coronary artery bypass graft or astent). In another aspect, when the ECM suspension is injected at thetarget site after the inflammatory response of the subject hasdecreased, it is contemplated that the ECM suspension should be injectedat the target site after the acute phase of the myocardial infarction,during which negatively remodeling and scar tissue formation occur. Invarious aspects, it is contemplated that, following injection of adisclosed ECM suspension on or within the heart of a subject, the ECMsuspension will not disperse but will instead attract stem cells to thetarget site, thereby promoting desired positive remodeling of the heart.As used herein, the term “positive remodeling” refers to beneficialregeneration and/or restructuring of damaged heart tissue; such positiveremodeling promotes growth of new cells while preserving thefunctionality of the heart and preventing formation of scar tissue.

Sterilization and Decellularization of the ECM Compositions

Optionally, it is contemplated that the disclosed extracellular matrixcompositions can be sterilized using a known sterilization system, suchas, for example and without limitation, the system described in U.S.Pat. No. 7,108,832, assigned to NovaSterilis, Inc., which patent isexpressly incorporated herein by reference in its entirety. Thus, insome aspects, the system used to perform the disclosed methods cancomprise a standard compressed storage cylinder and a standard aircompressor used in operative association with a booster (e.g., a HaskelBooster AGT 7/30). In other aspects, the air compressor and booster canbe replaced with a single compressor. In exemplary aspects, thecompressed storage cylinder can be configured to receive carbon dioxide,and the booster can be a carbon dioxide booster.

The system can further comprise an inlet port, which allows one or moreadditives contained in a reservoir to be added to a reactor vesselthrough a valve and an additive line. As used herein, the term “reactorvessel” refers to any container having an interior space that isconfigured to receive an ECM material and permit exposure of the ECMmaterial to one or more sterilants and additives, as disclosed herein.In exemplary aspects, the reactor vessel can be, without limitation, abasket, a bucket, a barrel, a box, a pouch, and other known containers.It is contemplated that the reactor vessel can be re-sealable. In oneaspect, it is contemplated that the reactor vessel can be a syringe thatis filled with an ECM material. In an exemplary aspect, the reactorvessel can be a pouch comprising Tyvek® packaging (E.I. du Pont deNemours and Company).

It is contemplated that a selected primary sterilant, such as, forexample and without limitation, carbon dioxide, can be introduced to thereactor vessel from a header line via a valve and a supply line. It isfurther contemplated that a filter, such as, for example and withoutlimitation, a 0.5 μm filter, can be provided in the supply line toprevent escape of material from the vessel. In exemplary aspects, apressure gauge can be provided downstream of a shut-off valve in theheader line to allow the pressure to be visually monitored. A checkvalve can be provided in the header line upstream of the valve toprevent reverse fluid flow into the booster. In order to prevent anoverpressure condition existing in the header line, a pressure reliefvalve can optionally be provided.

In one aspect, depressurization of the reactor vessel can beaccomplished using an outlet line and a valve in communication with thereactor vessel. In this aspect, it is contemplated that thedepressurized fluid can exit the vessel via the supply line, be filteredby a filter unit, and then be directed to a separator, where filteredfluid, such as carbon dioxide, can be exhausted via an exhaust line. Itis further contemplated that valves can be incorporated into the variouslines of the apparatus to permit fluid isolation of upstream components.

In one exemplary aspect, the reactor vessel can comprise stainlesssteel, such as, for example and without limitation, 316 gauge stainlesssteel. In another exemplary aspect, the reactor vessel can have a totalinternal volume sufficient to accommodate the materials beingsterilized, either on a laboratory or commercial scale. For example, itis contemplated that the reactor vessel can have a length of about 8inches, an inner diameter of about 2.5 inches, and an internal volume ofabout 600 mL. In additional aspects, the reactor vessel can comprise avibrator, a temperature control unit, and a mechanical stirring systemcomprising an impeller and a magnetic driver. In one optional aspect, itis contemplated that the reactor vessel can contain a basket comprising316 gauge stainless steel. In this aspect, it is contemplated that thebasket can be configured to hold materials to be sterilized while alsoprotecting the impeller and directing the primary sterilant in apredetermined manner.

It is contemplated that the reactor vessel can be operated at a constantpressure or under continual pressurization and depressurization(pressure cycling) conditions without material losses due to splashingor turbulence and without contamination of pressure lines viaback-diffusion. It is further contemplated that the valves within thesystem can permit easy isolation and removal of the reactor vessel fromthe other components of the system. In one aspect, the top of thereactor vessel can be removed when depressurized to allow access to theinterior space of the reactor vessel.

Optionally, the system can comprise a temperature control unit thatpermits a user to adjustably control the temperature within the reactorvessel.

In use, the disclosed apparatus can be employed in a method of producinga sterilized, acellular ECM composition, such as disclosed herein.However, it is understood that the disclosed apparatus is merelyexemplary, and that any apparatus capable of performing the disclosedmethod steps can be employed to produce the sterilized, acellular ECMcomposition. Thus, the claimed method is in no way limited to aparticular apparatus.

It is contemplated that significant reductions in colony-forming units(CFUs) can be achieved in accordance with the disclosed methods bysubjecting an isolated ECM material to sterilization temperature andpressure conditions using a primary sterilant. Optionally, it iscontemplated that the primary sterilant can be combined with one or moresecondary sterilants to achieve desired sterilization. Optionally, it isfurther contemplated that selected additives can be incorporated into anECM material to impart desired characteristics to the resulting ECMcomposition. It is still further contemplated that the disclosed methodscan be employed to produce sterilized, acellular ECM compositions forimplantation within the body of a subject.

As described herein, the disclosed methods make use of rapiddepressurization of an isolated ECM material to render the ECM materialacellular. This rapid depressurization of the ECM material occurs atdepressurization rates that are significantly higher than thedepressurization rates applied in previously known methods. In additionto rendering acellular the ECM material as described herein, the rapiddepressurization of the ECM material also can be used to enhance theincorporation of desired sterilants and additives into the ECM material.Further, it is contemplated that the rapid depressurization of the ECMmaterial can render the ECM material acellular while also improvingretention of native growth factors, as compared to previously knowndecellularization methods. Still further, it is contemplated that therapid depressurization of the ECM material can be used to improveretention of the tensile strength of the ECM material, as compared topreviously known decellularization methods.

The disclosed methods not only do not significantly weaken themechanical strength and bioptric properties of the ECM compositions, butalso the methods are more effective in decellularizing the ECMcompositions and in enhancing the incorporation of various additivesinto the ECM compositions. Thus, the disclosed sterilization anddecellularization methods provide ECM compositions that are moredecellularized and have a greater capacity to incorporate and thendeliver more additives than ECM compositions known in the art. Moreover,the disclosed sterilization and decellularization methods provide ECMcompositions that have greater amounts and/or concentrations of retainednative growth factors and that have greater tensile strength thansterilized and decellularized ECM compositions known in the art.

In exemplary aspects, the primary sterilant can be carbon dioxide at ornear its supercritical pressure and temperature conditions. However, itis contemplated that any conventional sterilant, including, for example,gas, liquid, or powder sterilants that will not interfere with thenative properties of the ECM material, can be used as the primarysterilant.

In one exemplary aspect, the disclosed sterilization process can bepracticed using carbon dioxide as a primary sterilant at pressuresranging from about 1,000 to about 3,500 psi and at temperatures rangingfrom about 25° C. to about 60° C. More preferably, when supercriticalcarbon dioxide is used, it is contemplated that the sterilizationprocess can use carbon dioxide as a primary sterilant at pressures at orabove 1,071 psi and at temperatures at or above 31.1° C. In this aspect,the ECM material to be sterilized can be subjected to carbon dioxide ator near such pressure and temperature conditions for times ranging fromabout 10 minutes to about 24 hours, more preferably from about 15minutes to about 18 hours, and most preferably, from about 20 minutes toabout 12 hours. Preferably, the carbon dioxide employed in the disclosedsystems and methods can be pure or, alternatively, contain only traceamounts of other gases that do not impair the sterilization propertiesof the carbon dioxide. For ease of further discussion below, the term“supercritical carbon dioxide” will be used, but it will be understoodthat such a term is non-limiting in that carbon dioxide within thepressure and temperature ranges as noted above can be employedsatisfactorily in the practice of the disclosed methods. Within thedisclosed pressure and temperature ranges, it is contemplated that thecarbon dioxide can be presented to the ECM material in a gas, liquid,fluid or plasma form.

The secondary sterilants employed in the disclosed methods can, in someaspects, include chemical sterilants, such as, for example and withoutlimitation, peroxides and/or carboxylic acids. Preferred carboxylicacids include alkanecarboxylic acids and/or alkanepercarboxylic acids,each of which can optionally be substituted at the alpha carbon with oneor more electron-withdrawing substituents, such as halogen, oxygen andnitrogen groups. Exemplary species of chemical sterilants employed inthe practice of the disclosed methods include, for example and withoutlimitation, hydrogen peroxide (H₂O₂), acetic acid (AcA), peracetic acid(PAA), trifluoroacetic acid (TFA), and mixtures thereof. In oneexemplary aspect, the chemical sterilants can include Sporeclenz®sterilant, which is a mixture comprising acetic acid, hydrogen peroxide,and peracetic acid.

It is contemplated that the secondary sterilants can be employed in asterilization-enhancing effective amount of at least about 0.001 vol. %and greater, based on the total volume of the primary sterilant. It isfurther contemplated that the amount of secondary sterilant can bedependent upon the particular secondary sterilant that is employed.Thus, for example, it is contemplated that peracetic acid can be presentin relatively small amounts of about 0.005 vol. % and greater, whileacetic acid can be employed in amounts of about 1.0 vol. % and greater.Thus, it is contemplated that the concentration of the secondarysterilants can range from about 0.001 vol. % to about 2.0 vol. % and cantypically be used as disclosed herein to achieve asterilization-enhancing effect in combination with the disclosed primarysterilants, such as, for example and without limitation, supercriticalcarbon dioxide.

In one aspect, the method of producing a sterilized, acellular ECMcomposition can comprise harvesting a selected tissue from a mammal andrinsing the selected tissue in sterile saline or other biocompatibleliquid, including, for example and without limitation, Ringer's solutionor a balanced biological salt solution. In this aspect, the selectedtissue can be, for example and without limitation, stomach tissue (e.g.,stomach submucosa (SS)), small intestinal tissue (e.g., small intestinalsubmucosa (SIS)), large intestinal tissue, bladder tissue (e.g., urinarybladder submucosa (UBS)), liver tissue (e.g., liver basement membrane(LBM)), heart tissue (e.g., pericardium, epicardium, endocardium,myocardium), lung tissue, kidney tissue, pancreatic tissue, prostatetissue, mesothelial tissue, fetal tissue, a placenta, a ureter, veins,arteries, heart valves with or without their attached vessels, tissuesurrounding the roots of developing teeth, and tissue surroundinggrowing bone. In another aspect, the method can comprise freezing theselected tissue for a period ranging from about 12 to about 36 hours,more preferably, from about 18 to about 30 hours, and most preferably,from about 22 to about 26 hours. For example, it is contemplated thatthe period during which the selected tissue is frozen can be 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34hours, 35 hours, 36 hours, and any other period of time falling betweenthe preceding values. In an additional aspect, the method can comprisethawing the selected tissue in cold hypotonic tris buffer. Optionally,in this aspect, the method can comprise thawing the selected tissue incold hypotonic tris buffer on ice with 5 mM ethylenediaminetetraaceticacid (EDTA). In exemplary aspects, it is contemplated that the steps offreezing and thawing the selected tissue can be cyclically repeated upto six times.

In another aspect, the method can comprise isolating an ECM materialfrom the selected tissue. In this aspect, the ECM material can be anymaterial comprising known extracellular matrix components, including,for example and without limitation, stomach tissue (e.g., stomachsubmucosa (SS)), small intestinal tissue (e.g., small intestinalsubmucosa (SIS)), large intestinal tissue, bladder tissue (e.g., urinarybladder submucosa (UBS)), liver tissue (e.g., liver basement membrane(LBM)), heart tissue (e.g., pericardium, epicardium, endocardium,myocardium), lung tissue, kidney tissue, pancreatic tissue, prostatetissue, mesothelial tissue, fetal tissue, a placenta, a ureter, veins,arteries, heart valves with or without their attached vessels, tissuesurrounding the roots of developing teeth, and tissue surroundinggrowing bone. In one exemplary, non-limiting aspect, the step ofisolating an ECM material can comprise isolating SIS from a mammaliantissue source. In this aspect, the method can comprise: incising a wallof a small intestine along a path that is substantially parallel to thelongitudinal axis of the small intestine; opening the small intestinealong the path of the incision such that the small intestine lies flaton a surface; rinsing the small intestine with sterile saline or otherbiocompatible fluid; mechanically stripping the SIS of the smallintestine from the surrounding smooth muscle and serosal layers and fromthe tunica mucosa, leaving essentially the submucosal and basementmembrane layers. However, it is contemplated that the ECM material canbe isolated using any conventional technique, including those describedin: U.S. Pat. No. 4,902,508; U.S. Pat. No. 5,275,826; U.S. Pat. No.5,281,422; U.S. Pat. No. 5,554,389; U.S. Pat. No. 6,579,538; U.S. Pat.No. 6,933,326; U.S. Pat. No. 7,033,611; Voytik-Harbin et al.,“Identification of Extractable Growth Factors from Small IntestinalSubmucosa,” J. Cell. Biochem., Vol. 67, pp. 478-491 (1997); Hodde etal., “Virus Safety of a Porcine-Derived Medical Device: Evaluation of aViral Inactivation Method,” Biotech. & Bioeng., Vol. 79, No. 2, pp.211-216 (2001); Badylak et al., “The Extracellular Matrix as a Scaffoldfor Tissue Reconstruction,” Cell & Developmental Biology, Vol. 13, pp.377-383 (2002); Robinson et al., “Extracelular Matrix Scaffold forCardiac Repair,” Circulation, Vol. 112, pp. I-135-I-143 (2005); Hodde etal., “Effects of Sterilization on an Extracellular Matrix Scaffold: PartI. Composition and Matrix Architecture,” J. Mater. Sci.: Mater. Med.,Vol. 18, pp. 537-543 (2007); and Hodde et al., “Effects of Sterilizationon an Extracellular Matrix Scaffold: Part II. Bioactivity and MatrixInteraction,” J. Mater. Sci.: Mater. Med., Vol. 18, pp. 545-550 (2007),each of which is expressly incorporated herein by reference in itsentirety.

In an additional aspect, the method can comprise incubating the isolatedECM material for 24 to 48 hours in 0.5-1% Triton X-100/0.5-1%Deoxycholic acid with 5 mM EDTA in Dulbecco's Phosphate Buffered Saline(DPBS) (Lonza Walkersville, Inc.). In this aspect, it is contemplatedthat flat or sheet-like ECM materials, such as stomach submucosa (SS),small intestinal submucosa (SIS), and urinary bladder submucosa (UBS),can be incubated in a stretched configuration. It is furthercontemplated that ECM material conduits or other lumenal ECM materials,such as ureters, arteries, veins, and tubular SIS, can be perfused withthe various disclosed solutions through soaking and by use of aperistaltic pump.

In a further aspect, after incubation, the method can comprise rinsingthe ECM material with DPBS. In this aspect, it is contemplated that thestep of rinsing the ECM material can comprise rinsing the ECM materialup to six times, including one, two, three, four, five, or six times,with each rinse lasting for about thirty minutes. In an exemplaryaspect, it is contemplated that the step of rinsing the ECM material cancomprise rinsing the ECM material three times, with each rinse lastingfor about thirty minutes.

Optionally, in exemplary aspects, the method can further comprise asecond incubation procedure. In these aspects, the second incubationprocedure can comprise incubating the ECM material in isotonic trisbuffer containing 10-50 μg/mL of RNAase/0.2-0.5 μg/mL DNAase with 5 mMEDTA. It is contemplated that the step of incubating the ECM material inisotonic tris buffer can be performed at a temperature of about 37° C.,substantially corresponding to the temperature of a human body. It isfurther contemplated that the step of incubating the ECM material inisotonic tris buffer can be performed for a period ranging from about 30minutes to about 24 hours, more preferably, from about 1 hour to about18 hours, and most preferably, from about 2 hours to about 12 hours. Inan additional aspect, the second incubation procedure can furthercomprise rinsing the ECM material with DPBS. In this aspect, it iscontemplated that the step of rinsing the ECM material can compriserinsing the ECM material three times, with each rinse lasting for aboutthirty minutes.

In yet another aspect, whether or not the second incubation procedure isperformed, the method can comprise storing the ECM material at atemperature ranging from about 1° C. to about 10° C., more preferably,from about 2° C. to about 6° C., and, most preferably, from about 3° C.to about 5° C. In an exemplary aspect, the ECM material can be stored at4° C.

In an additional aspect, the method can comprise introducing the ECMmaterial into the interior space of a reactor vessel. Optionally, inthis aspect, one or more secondary sterilants from the reservoir can beadded into the interior space of the reactor vessel along with the ECMmaterial. In these aspects, it is contemplated that the one or moresecondary sterilants from the reservoir can be added into the interiorspace of the reactor vessel before, after, or contemporaneously with theECM material. It is further contemplated that the temperature controlunit can be selectively adjusted to produce a desired temperature withinthe interior space of the reactor vessel. In a further aspect, themethod can comprise equilibrating the pressure within the reactor vesseland the pressure within the storage cylinder. For example, in thisaspect, it is contemplated that the pressure within the reactor vesseland the pressure within the storage cylinder can be substantially equalto atmospheric pressure. In yet another aspect, after equilibration ofthe pressures within the apparatus, the method can comprise operatingthe magnetic driver to activate the impeller of the reactor vessel. Instill a further aspect, the method can comprise selectively introducingthe primary sterilant from the storage cylinder into the reactor vesseluntil a desired pressure within the reactor vessel is achieved. In thisaspect, it is contemplated that the step of selectively introducing theprimary sterilant into the reactor vessel can comprise selectivelyactivating the air compressor and the booster to increase flow of theprimary sterilant into the reactor vessel. In exemplary aspects, the aircompressor and booster can be activated to subject the ECM material tosupercritical pressures and temperatures, such as, for example andwithout limitation, the pressures and temperatures necessary to producesupercritical carbon dioxide, for a time period ranging from about 20minutes to about 60 minutes.

In a further aspect, the method can comprise rapidly depressurizing thereactor vessel. In this aspect, a predetermined amount of primarysterilant, such as, for example and without limitation, supercriticalcarbon dioxide, can be released from the reactor vessel through thedepressurization line. It is contemplated that the primary sterilant canbe released from the reactor vessel through opening of the valve coupledto the reactor vessel to thereby rapidly reduce the pressure within thereactor vessel. As used herein, the term “rapid depressurization” refersto depressurization of the reactor vessel at a rate greater than orequal to 400 psi/min. For example, it is contemplated that the reactorvessel can be depressurized at a depressurization rate ranging fromabout 2.9 MPa/min. to about 18.0 MPa/min. (about 420 psi/min. to about2,610 psi/min.), more preferably from about 5.0 MPa/min. to about 10.0MPa/min. (725 psi/min. to about 1,450 psi/min.), and, most preferably,from about 7.0 MPa/min. to about 8.0 MPa/min. (about 1,015 psi/min. toabout 1,160 psi/min.). Thus, these rapid depressurizations aresignificantly greater than the 300 psi/min. depressurization ratedisclosed in U.S. Pat. No. 7,108,832. Without being bound by anyparticular theory, it is believed that the disclosed rapiddepressurization rates increase the level of decellularization achievedin the ECM material. For example, the rapid depressurization of adisclosed ECM material can lead to levels of decellularization in theECM material of greater than about 96%, including 96.1%, 96.2%, 96.3%,96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%,97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%,98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and 100.0%.

In exemplary aspects, the method can further comprise the step ofincorporating one or more additives into the ECM material. In theseaspects, it is contemplated that the one or more additives can beprovided in either a powder or a liquid form. In one optional aspect,the step of incorporating the one or more additives can compriseintroducing the one or more additives into the reactor vessel during thestep of rapidly depressurizing the reactor vessel. In this aspect, it iscontemplated that the introduction of the one or more additives can becharacterized as a conventional foaming process. In another optionalaspect, the step of incorporating the one or more additives can compriseintroducing the one or more additives into the reactor vessel after thestep of rapidly depressurizing the reactor vessel. In this aspect, it iscontemplated that the one or more additives can be added to the ECMmaterial after the rapid depressurization of the reactor vessel hascaused the ECM material to swell and/or expand, thereby permittingenhanced penetration of the additives into the ECM material. It isfurther contemplated that, in an exemplary aspect, the one or moreadditives can be added to the ECM material within about thirty minutesafter the rapid depressurization of the reactor vessel. In a furtheroptional aspect, the step of incorporating the one or more additives cancomprise introducing the one or more additives into the reactor vesselboth during and after the step of rapidly depressurizing the reactorvessel. In this aspect, it is contemplated that the one or moreadditives can be released into the reactor vessel in both a quick mannerand a slow, extended manner. In still a further optional aspect, thestep of incorporating the one or more additives can comprise introducingthe one or more additives into the reactor vessel before the step ofrapidly depressurizing the reactor vessel.

The disclosed additives can be incorporated into the ECM material toimpart selected properties to the resulting sterilized, acellular ECMcomposition. Thus, it is contemplated that the one or more additives canbe selected to replace or supplement components of the ECM material thatare lost during processing of the ECM material as described herein. Forexample, and as described below, the one or more additives can comprisegrowth factors, cytokines, proteoglycans, glycosaminoglycans (GAGS),proteins, peptides, nucleic acids, small molecules, drugs, or cells. Itis further contemplated that the one or more additives can be selectedto incorporate non-native components into the ECM material. For example,the one or more additives can comprise, for example and withoutlimitation, growth factors for recruiting stem cells, angiogeniccytokines, and anti-inflammatory cytokines. It is still furthercontemplated that the one or more additives can be pharmaceuticalagents, such as statins, corticosteroids, non-steroidalanti-inflammatory drugs, anti-inflammatory compounds, anti-arrhythmicagents, and the like. It is still further contemplated that the one ormore additives can be nanoparticles, such as, for example and withoutlimitation, silver nanoparticles, gold nanoparticles, platinumnanoparticles, iridium nanoparticles, rhodium nanoparticles, palladiumnanoparticles, copper nanoparticles, zinc nanoparticles, and othermetallic nanoparticles. It is still further contemplated that the one ormore additives can be metallic compounds. In one exemplary aspect, theone or more additives can be selected to pharmaceutically suppress theimmune response of a subject following implantation of the resulting ECMcomposition into the body of a subject.

In one aspect, the one or more additives can comprise one or more growthfactors, including, for example and without limitation, transforminggrowth factor-β-1, -2, or -3 (TGF-β-1, -2, or -3), fibroblast growthfactor-2 (FGF-2), also known as basic fibroblast growth factor (bFGF),vascular endothelial growth factor (VEGF), placental growth factor(PGF), connective tissue growth factor (CTGF), hepatocyte growth factor(HGF), Insulin-like growth factor (IGF), macrophage colony stimulatingfactor (M-CSF), platelet derived growth factor (PDGF), epidermal growthfactor (EGF), and transforming growth factor-α (TGF-α).

In another aspect, the one or more additives can comprise one or morecytokines, including, for example and without limitation, stem cellfactor (SCF), stromal cell-derived factor-1 (SDF-1), granulocytemacrophage colony-stimulating factor (GM-CSF), interferon gamma(IFN-gamma), Interleukin-3, Interleukin-4, Interleukin-10,Interleukin-13, Leukemia inhibitory factor (LIF), amphiregulin,thrombospondin 1, thrombospondin 2, thrombospondin 3, thrombospondin 4,thrombospondin 5, and angiotensin converting enzyme (ACE).

In an additional aspect, the one or more additives can comprise one ormore proteoglycans, including, for example and without limitation,heparan sulfate proteoglycans, betaglycan, syndecan, decorin, aggrecan,biglycan, fibromodulin, keratocan, lumican, epiphycan, perlecan, agrin,testican, syndecan, glypican, serglycin, selectin, lectican, versican,neurocan, and brevican.

In a further aspect, the one or more additives can comprise one or moreglycosaminoglycans, including, for example and without limitation,heparan sulfate, hyaluronic acid, heparin, chondroitin sulfate B(dermatan sulfate), and chondroitin sulfate A.

In still a further aspect, the one or more additives can comprise one ormore proteins, peptides, or nucleic acids, including, for example andwithout limitation, collagens, elastin, vitronectin, versican, laminin,fibronectin, fibrillin-1, fibrillin-2, plasminogen, small leucine-richproteins, cell-surface associated protein, cell adhesion molecules(CAMs), a matrikine, a matrix metalloproteinase (MMP), a cadherin, animmunoglobin, a multiplexin, cytoplasmic domain-44 (CD-44), amyloidprecursor protein, tenascin, nidogen/entactin, fibulin I, fibulin II,integrins, transmembrane molecules, and osteopontin.

In yet another aspect, the one or more additives can comprise one ormore pharmaceutical agents, including, for example and withoutlimitation, statin drugs, for example, cerevastatin, atorvastatin,fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin,rosuvastatin, and simvastatin; corticosteroids; non-steroidalanti-inflammatory drugs; anti-inflammatory compounds; anti-arrhythmicagents; antimicrobials; antibiotics; and the like.

In exemplary aspects, the steps of introducing the one or more additivesinto the reactor vessel can comprise opening the valve to allow the oneor more additives to flow from the reservoir into the inlet port. Priorto pressurization, it is contemplated that the one or more additives canbe introduced directly into the reactor vessel prior to sealing and/orvia the inlet port.

It is contemplated that the disclosed rapid depressurization andrepressurization of the reactor vessel, with or without the addition ofthe one or more additives, can be repeated for any desired number ofcycles. It is further contemplated that the cycles of depressurizationand repressurization, as well as the introduction of the primarysterilants and/or secondary sterilants and/or additives, can beautomatically controlled via a controller that is configured toselectively open and/or close the various valves of the system toachieve desired pressure conditions and cycles.

In some aspects, the disclosed methods can further comprise the step ofagitating the contents of the reactor vessel. In these aspects, it iscontemplated that the step of agitating the contents of the reactorvessel can comprise periodically agitating the contents of the reactorvessel using a vibrator. It is further contemplated that the agitationof the reactor vessel can be intermittent, continual, or continuous. Inexemplary aspects, the step of agitating the contents of the reactorvessel can occur during the step of introducing the primary sterilantinto the reactor vessel. It is contemplated that the agitation of thecontents of the reactor vessel can enhance the mass transfer of thesterilants and/or additives by eliminating voids in the fluids withinthe reactor vessel to provide for more complete contact between the ECMmaterial and the sterilants and/or additives. It is further contemplatedthat the step of agitating the contents of the reactor vessel cancomprise selectively adjusting the intensity and duration of agitationso as to optimize sterilization times, temperatures, andpressurization/depressurization cycles.

In a further aspect, after the sterilization and decellularization ofthe ECM material is complete, the method can further comprisedepressurizing the reactor vessel and deactivating the magnetic drive soas to cease movement of the stirring impeller. Finally, the method cancomprise the step of removing the resulting sterilized, acellular ECMcomposition through the top of the reactor vessel.

It is contemplated that the duration of the disclosed steps, as well asthe temperatures and pressures associated with the disclosed steps, canbe selectively varied to account for variations in the characteristicsof the ECM material. For example, when the ECM material is amulti-laminate structure, has an increased thickness, or is positionedwithin a syringe, it is contemplated that the duration of the disclosedsteps can be increased.

In one optional aspect, in order to make the sterilized, acellular ECMcomposition into a particulate form, the method can comprise cutting theECM composition into pieces having desired lengths. In another aspect,the method can optionally comprise freeze-drying the pieces of the ECMcomposition. In an additional aspect, the method can optionally comprisegrinding the frozen, hydrated pieces of the ECM composition and thenpassing the pieces of the ECM composition through a sizer screen untilECM particulate of a desired size is isolated. In a further optionalaspect, the method can comprise rehydrating the ECM particulate withsterile saline or other sterile, biocompatible fluid to form an ECMsuspension, as described herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1 Retrospective Evaluation of New Onset Postoperative AtrialFibrillation in Patients Receiving the CorMatrix® ECM™

A retrospective, multi-center, two-arm, chart review was conducted inwhich the CorMatrix® ECM™ was utilized. The objective of thisretrospective trial was to assess whether utilization of the CorMatrix®ECM™ to reconstruct the normal pericardial barrier can result in a lowerrate of new onset postoperative atrial fibrillation as compared topatients who did not undergo pericardial closure.

CorMatrix® ECM™ can be used for the reconstruction and repair of thepericardium following open heart surgery. Intact, the pericardiumprovides passive restraint to the heart preventing over dilation andhelping to modulate abrupt volumetric changes. By reconstructing thepericardium with the CorMatrix® ECM™, the natural pericardial restraintcan be restored. The purpose of this retrospective clinical trial was toassess if there is a reduction observed in new onset postoperativeatrial fibrillation by analyzing patients who had their nativepericardium reconstructed with the CorMatrix® ECM™ as compared to thosewho did not undergo pericardial closure following isolated coronaryartery bypass graft (CABG) procedures.

The CorMatrix® ECM™ was supplied in four-ply sheets of variousdimensions, which can be cut to size as the physician deems necessaryfor the procedure.

The definition of new onset postoperative atrial fibrillation used forthis retrospective study is based on the definition used in the Societyof Thoracic Surgeons (STS) Adult Cardiac Surgery Database 2007. Thedefinition is as follows: “Indicate whether the patient had a new onsetof Atrial Fibrillation/Flutter (AF) requiring treatment. Does notinclude recurrence of AF which had been present preoperatively. DO NOTinclude patients that had preoperative atrial fibrillation (treated ornontreated). The event must be of new origin.

All patients were required to meet the following inclusion criteria inorder to be included as part of this retrospective clinical trial: thiscardiac operation was the subject's first or primary cardiac operation,and the subject must have undergone an isolated CABG procedure.

Patients were not included as part of this retrospective clinical trialif one or more of the following exclusion criteria are met: priorhistory of atrial fibrillation, prior history of open heart surgery,prior history of pericarditis, prior history of amiodarone in the pastsix months, and concomitant valve surgery planned.

Patients who had their native pericardium reconstructed with theCorMatrix® ECM™ had a statistically significant decrease in theincidence of A-fib as compared to those who did not undergo pericardialclosure following isolated CABG procedures. The usual incidence of A-fibis around 25%. For these studies, the A-fib incidence was between 4% and8% (1/25 and 4/52).

Example 2 Modulation of Cardiac Remodeling with Acellular MatrixEmulsion is Associated with Myofibroblast Proliferation and AngiogenesisVia Recruiting C-Kit Positive Cells after Myocardial Infarction

Degradation of native extracellular matrix (ECM) has been associatedwith maladaptive cardiac remodeling after infarction. As shown herein,xenogeneic acellular matrix emulsion injected into infarcted myocardiumpromoted myofibroblast proliferation and angiogenesis by recruiting hostc-kit positive cells.

Sixty-four rats were subjected to 45 minutes regional ischemia followedby 3, 7, 21 and 42 days of reperfusion. Histological examination wasperformed by immunohistological staining, and cardiac function wasanalyzed using echocardiography. ECM emulsion (30-50 μl) was injectedinto the area at risk myocardium after reperfusion, and localization ofthe emulsion was confirmed with Masson Trichome staining. At 7 days ofreperfusion, the population of c-kit positive cells within the emulsionarea increased significantly relative to the control (32±0.6* vs.15±3/1000 nuclei), consistent with significantly enhanced expression of31 kDa stem cell factor detected by Western blotting. Along with thischange, myofibroblasts accumulated in the emulsion region to asignificant extent compared to the control (59±8* vs. 30±3/HPF). Strongimmunoreactivity of VEGF was observed in the emulsion area andangiogenesis was significantly enhanced relative to the control,evidenced by increased density of a-smooth muscle actin-positive vessels(70±10* vs. 20±4/HPF) and vWF-positive vessels (95±14* vs. 34±8/HPF),respectively. At 42 days of reperfusion, echocardiography showedimprovements in end-systolic volume (0.3±0.1* vs. 0.6±0.3 ml)),fractional shortening (33±5* vs. 24±6%) and ejection fraction (67±6* vs.53±10%) in the emulsion group. The wall thickness of the infarctedmiddle anterior septum in the emulsion group was also significantlygreater than that in the Control (0.19±0.02* vs. 0.15±0.02 cm).

Intramyocardial injection of an acellular extracellular matrix emulsioninto the ischemic/reperfused myocardium attenuated maladaptive cardiacremodeling and preserved cardiac function, potentially mediated byenhanced myofibroblast proliferation and angiogenesis via recruitingc-kit positive cells. * p<0.05 emulsion vs. control.

Example 3

In exemplary applications of the disclosed sterilization anddecellularization methods, selected tissues were harvested and rinsed insterile saline. The selected tissues were then frozen for 24 hours. Thefrozen tissues were thawed in cold hypotonic tris buffer on ice with 5mM ethylenediaminetetraacetic acid (EDTA). An extracellular matrixmaterial was then isolated from each selected tissue, as describedherein.

The isolated extracellular matrix materials were incubated for 24 to 48hours in 0.5-1% Triton X-100/0.5-1% Deoxycholic acid with 5 mM EDTA inDulbecco's Phosphate Buffered Saline (DPBS) (Lonza Walkersville, Inc.).Flat extracellular matrix materials, such as stomach submucosa (SS),small intestinal submucosa (SIS), and bladder submucosa (UBS), wereincubated in a stretched configuration. Tubular extracellular matrixmaterials, such as ureters, arteries, veins, and tubular SIS, wereperfused with the solutions through soaking and by use of a peristalticpump.

After incubation, each extracellular matrix material was rinsed threetimes with DPBS. Each rinsing with DPBS lasted 30 minutes. Someextracellular matrix materials were then incubated for 2 to 12 hours at37° C. in isotonic tris buffer containing 10-50 μg/mL of RNAse/0.2-0.5μg/mL DNAse with 5 mM EDTA. Following this incubation step, theextracellular matrix materials were again rinsed three times with DPBS.Each rinsing with DPBS lasted 30 minutes. The extracellular matrixmaterials were stored at 4° C.

Within 48 hours of storage, the extracellular matrix materials wereprocessed in supercritical carbon dioxide as disclosed herein for 20-60minutes at temperatures at or greater than 31.1° C. and pressures at orgreater than 1,071 psi. After this sterilization step, the extracellularmatrix materials were rapidly depressurized at a rate of 2.7 MPa/10 sec.(391.6 psi/10 sec.) for a minute and 19 seconds. During this time, thepressure applied to the extracellular matrix materials rapidly decreasedfrom 9.9 MPa to 0.69 MPa.

The extracellular matrix materials were then processed in supercriticalcarbon dioxide and peracetic acid (PAA) as disclosed herein for 30minutes to 6 hours to achieve terminal sterilization. In this processingstep, the pressure applied to the extracellular matrix materials wasincreased to 9.9 MPa. The resulting sterilized, acellular extracellularmatrix materials were then packaged in Tyvek® (E.I. du Pont de Nemours &Company) pouches that were sealed within plastic pouches to preventfluid leakage.

Table 1 summarizes the sterilization and decellularization of porcineureter, bovine pericardium, and porcine mesothelium.

TABLE 1 RNAse/ Triton TX-100/ DNAse Supercritical X-100 DeoxycholicDeoxy incuba- CO₂/PAA Material Conc. Acid Conc. incubation tion timePorcine 0.5% 0.5% 24 hours 2 hours 120 minutes ureters Bovine 0.5% 0.5%24 hours 2 hours 180 minutes pericardium Porcine 0.5% 0.5% 24 hours 2hours 120 minutes mesothelium

Example 4

The DNA content of ECM material samples was measured as an indicator ofdecellularization of the respective ECM material samples using varioussterilization and decellularization techniques. The measured DNA contentwas evaluated with a pico green assay in which DNA was labeled with afluorescent label that was detected with a spectrophotometer. Themeasured DNA content was normalized by the dry weight of the samples.DNA content was measured and evaluated for the following treatmentgroups: (1) Lyophilized, non-sterile SIS; (2) Ethylene Oxide(EtO)-sterilized SIS; (3) Lyophilized, non-sterile SIS that wassterilized through a 60 minute treatment with PAA and supercritical CO₂,as disclosed herein; (4) Lyophilized, non-sterile SIS that wassterilized through a 20 minute treatment with PAA and supercritical CO₂,as disclosed herein; and (5) Raw, unprocessed SIS.

FIG. 1 shows the total DNA content for the respective samples, asnormalized by dry weight. FIG. 2 shows the percent of DNA that wasremoved from each respective sample, as compared to raw, unprocessedSIS. These results indicated that by sterilizing the non-sterile SISusing a 60 minute treatment with PAA and supercritical CO₂, as disclosedherein, over 96% of the DNA found in raw SIS was removed, as compared toonly 94% when the SIS was sterilized by EtO and only 93% when the SISwas not sterilized by any method.

Example 5

Ureters were processed with a gentle detergent (0.5% Triton X-100/0.5%Sodium Deoxycholate in 5 mM EDTA in DPBS) for 24 hours and then rinsedthree times in DPBS as disclosed herein. After this pretreatment, theureters were decellularized and sterilized using rapid depressurizationand treatment with PAA and supercritical CO₂, as disclosed herein.Hematoxylin and Eosin (H&E) Stains were prepared for one sample ureterat the following stages of treatment: (A) native ureter; (B) pretreatedureter; and (C) pretreated ureter with rapid depressurization andtreatment with PAA and supercritical CO₂, as disclosed herein. Thesestains indicated that DNA content was significantly reduced with rapiddepressurization.

Example 6

The growth factor content of ECM material samples was measured.Enzyme-linked immunosorbent (ELISA) assays were performed on the ECMmaterial samples to quantify the content of bFGF and the active form ofTGF-13 in each respective sample. The following treatment groups wereevaluated: (1) Lyophilized, non-sterile SIS; (2) Ethylene Oxide(EtO)-sterilized SIS; (3) Lyophilized, non-sterile SIS that wassterilized through a 60 minute treatment with PAA and supercritical CO₂,as disclosed herein; (4) Lyophilized, non-sterile SIS that wassterilized through a 20 minute treatment with PAA and supercritical CO₂,as disclosed herein; and (5) Raw, unprocessed SIS. The bFGF content andTGF-β content measurements were normalized by dry weight of eachrespective sample. These results are shown in FIGS. 3 and 4. Theseresults indicated that the concentration of both growth factors wasreduced by exposure to EtO. However, the concentration of the growthfactors was not affected by sterilization with PAA and supercriticalCO₂.

Example 7

Using the methods disclosed herein, supercritical CO₂ was used as aprimary sterilant and as a carrier for adding bFGF into SIS sheets.First, the respective SIS sheets were placed into Tyvek® pouches alongwith varying amounts of bFGF. The pouches were exposed to supercriticalCO₂ for 60 minutes at 9.6 MPa. The pouches were rapidly depressurized ata rate of 7.20 MPa/min. Samples were directly processed in 16 mL PAA insupercritical CO₂ for 20 minutes. The following treatment groups wereevaluated: (1) No bFGF added; (2) 5 μL bFGF added; and (3) 15 μL bFGFadded. Each 4 of bFGF contained 0.1 μg of bFGF. Thus, since each SISsheet weighed approximately 0.5 g, the maximum concentrations of bFGFfor the 5 μL and 15 μL groups were about 4170 pg/mg dry weight and about12,500 pg/mg dry weight, respectively. The bFGF content for these groupsis shown in FIG. 5, as measured with respect to the dry weight of therespective samples. These results indicated that the measuredconcentrations of bFGF did not reach the maximum concentrations and thatthe sample to which 15 μL of bFGF was added did not have a measuredconcentration of bFGF that was three times greater than the measuredconcentration of bFGF in the sample to which 5 μL of bFGF was added.

Example 8

The tensile strengths of two-ply SIS samples were measured. Thefollowing treatment groups were evaluated: (1) EtO Treatment; (2)PAA/supercritical CO₂ treatment for 20 minutes; (3) PAA/supercriticalCO₂ treatment for 60 minutes; and (4) PAA/supercritical CO₂ treatmentfor 120 minutes. The tensile strength test results are shown in FIG. 6.These results indicated that the SIS samples that were processed withPAA/supercritical CO₂ for 20 or 120 minutes, as disclosed herein, weresignificantly stronger than the SIS samples that were processed withEtO.

Example 9

Rapid depressurization was used following gentle detergent soaks orperfusion of the ECM materials listed in Table 2 (below) at the notedconcentrations and for the noted time periods. Tissues were harvestedand rinsed in saline. The tissues were frozen for at least 24 hours. Thetissues were thawed in cold hypotonic tris buffer on ice with 5 mM EDTA.The ECM of interest was isolated. For flat tissues (e.g., stomachsubmucosa, small intestine submucosa, and bladder submucosa), the tissuewas stretched on a tissue stretching device and incubated in solutionsin a stretched configuration. For tubular tissues (e.g., ureters,arteries, veins, and tubular SIS), the tissue was perfused withsolutions using a peristaltic pump and were soaked during incubation.The tissues were incubated for 2 to 24 hours in 0.5% Triton X-100/0.5%Deoxycholic acid with 5 mM EDTA in DPBS. The tissues were rinsed 3 timesfor 15-30 minutes each time in DPBS. The tissues were stored at 4° C.Within 48 hours of tissue storage, the tissues were processed insupercritical CO₂ for 20-120 minutes followed by rapid depressurization(RDP)(decrease in pressure from 9.9 MPa to 0.69 MPa in 1 min 19 sec,corresponding to a depressurization of 2.7 MPa/10 sec).

TABLE 2 Triton X-100 Deoxycholic TX-100/Deoxy Supercritical MaterialConc. Acid Conc. incubation CO₂ time Porcine 0.5% 0.5% 24 hours 60minutes ureters Bovine 0.5% 0.5% 24 hours 60 minutes pericardium Porcine0.5% 0.5%  2 hours 60 minutes mesothelium SIS 0.5% 0.5%  2 hours 60minutes

The results showed that supercritical CO₂ exposure followed by rapiddepressurization (SCCO₂+RDP) did aid in the removal of cell remnants andDNA while preserving growth factors in the ECMs.

Example 10

The growth factor content of various ECM compositions was analyzed usingbasic fibroblast growth factor (bFGF) as a representative growth factor.bFGF was selected because it is a prevalent growth factor in native ECMtissues. An enzyme-linked immunosorbent assay (ELISA, R&D Systems,Minneapolis, Minn.) was used to measure the bFGF content in thefollowing samples: (1) Unprocessed (Raw) SIS; (2) SIS after detergentsoak (TX-deoxy) only; (3) SIS after TX-deoxy and RDP (includes SCCO₂);(4) SIS after TX-deoxy, RDP, and PAA (SCCO₂ with PAA for sterilization);(5) SIS after TX-deoxy, and PAA; (6) SIS sterilized by EtO (supplied byCook Biotech, Inc.); and (7) non-sterile SIS (supplied by Cook Biotech,Inc.).

In these studies, SIS was used to compare an ECM composition processedwith and without RDP to SIS provided by Cook Biotech, Inc. Some of theprocessed SIS was also sterilized using the described SCCO₂+PAA methodafter decellularization. The measured growth factor content of therespective ECM compositions is shown in FIG. 7.

These results indicate that the rapid depressurization process was moreeffective than other decellularization processes at preserving the bFGFcontent and that the additional RDP processing to remove residual DNAand cell fragments results in only a small loss of bFGF. By comparison,the PAA sterilization process appeared to remove almost all of theremaining bFGF, even in the absence of RDP. Additionally, the rapiddepressurization process preserved more of the bFGF content in thenative SIS than the Cook decellularization methods. For purposes ofthese results, when the bFGF content was reduced, it is assumed that allother growth factor content was similarly reduced since the growthfactors are all bound to the ECM compositions in a similar manner.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. An ECM composition for treating a cardiacarrhythmia, comprising: an sterilized, acellular ECM scaffold material,said ECM scaffold material comprising mesothelial tissue, saidmesothelial tissue being sterilized and decellularized by introducingsaid mesothelial tissue into a reactor pressure vessel, introducing atleast a first liquid sterilant into said reactor pressure vessel at afirst controlled pressurization rate, pressurization said reactorpressure vessel at a pressure in the range of approximately 1000-3500psi, maintaining said mesothelial tissue in contact with said firstliquid sterilant for a first time period in the range of approximately10 min. to 24 hours, and depressurizing said reactor pressure vessel ata first depressurizing rate, said first depressurizing rate beinggreater than 400 psi/min.
 2. The ECM composition of claim 1, whereinsaid first liquid sterilant comprises supercritical carbon dioxide.
 3. Asterilized, acellular ECM composition for treating a cardiac arrhythmia,comprising: an ECM scaffold material and at least a first non-nativebiologically active agent, said ECM scaffold material comprisingmesothelial tissue, said mesothelial tissue being sterilized anddecellularized by introducing said mesothelial tissue into a reactorpressure vessel, introducing at least a first liquid sterilant into saidreactor pressure vessel at a first controlled pressurization rate,pressurization said reactor pressure vessel at a pressure in the rangeof approximately 1000-3500 psi, maintaining said mesothelial tissue incontact with said first liquid sterilant for a first time period in therange of approximately 10 min. to 24 hours, and depressurizing saidreactor pressure vessel at a first depressurizing rate, said firstdepressurizing rate being greater than 400 psi/min., said firstnon-native biologically active agent being introduced into saidmesothelial tissue during said depressurization of said reactor pressurevessel.
 4. The ECM composition of claim 3, wherein said first liquidsterilant comprises supercritical carbon dioxide.
 5. The ECM compositionof claim 3, wherein said first non-native biologically active agentcomprises a statin selected from the group consisting of lovastatin,simvastatin, atorvastatin, fluvastatin, pravastatin, rosuvastatin,cerivastatin and pitavastatin.
 6. The ECM composition of claim 3,wherein said first non-native biologically active agent comprises ananti-arrhythmic agent selected from the group consisting of quinidine,procainamide, disopyramide, lidocaine, phenyloin, mexiletine, flecamide,propafenone, moricizine, propranolol, esmolol, timolol, metoprolol,atenolol, amiodarone, sotalol, ibutilide, dofetilide, verapamil,diltiazem, adenosine and digoxin.
 7. The ECM composition of claim 3,wherein said first non-native biologically active agent comprises ananti-microbial.
 8. The ECM composition of claim 3, wherein said cardiacarrhythmia comprises atrial fibrillation.